Development of amyloglucosidase as a medicinal food or dietary supplement

ABSTRACT

Embodiments of the disclosure include particular amyloglucosidase (AMG) compositions formulated as a nutriceutical or medicinal food, for example. The AMG compositions are formulated at a specific dosage and/or are lacking in one or more toxins or have substantially reduced levels of toxin, such as deoxynivalenol (vomit toxin). The AMG compositions are provided to individuals in need thereof, such as an individual with or at risk for congenital sucrase isomaltase syndrome, functional bowel disorders, small bowel bacterial overgrowth, protein-calorie malnutrition (marasmus), radiochemotherapy-induced mucositis and/or short-gut syndrome.

This application is a national phase application under 35 U.S.C. § 371that claims priority to International Application No. PCT/US2019/027925filed Apr. 17, 2019, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/659,459, filed Apr. 18, 2018, both of which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

Embodiments of the present disclosure concern at least the fields ofcell biology, nutrition, biochemistry, molecular biology,gastroenterology, endocrinology, and medicine.

BACKGROUND

In health, the intestinal mucosal cells function to absorbmonosaccharides from hydrolyzed complex dietary carbohydrates and, whendamaged, are significantly impaired from completely hydrolyzingoligosaccharides during an illness that often results with chronicdiarrhea. The mucosal cells are dynamic and assimilate the luminaldigestive products and transfer nutrients to various endogenousmetabolic processes (Ravich and Bayless, 1983). At the earliest level,food digestion critically depends upon secretion of salivary andpancreatic α-amylase and cellular expression of several other apicaldigestive enzymes. First, dietary starch is ‘coarsely’ hydrolyzed in theduodenum and jejunum, by intraluminal pancreatic alpha-amylase torelease soluble maltotriose, oligosaccharides and alpha-limit dextrins(oligomers). These intermediate by-products of partial digestion are notdirectly absorbable and must undergo further enzymatic hydrolysis(cleavage) at the apical enterocyte surface to monosaccharides (glucose,fructose, and galactose) by expressed a cadre of disaccharidasesincluding sucrase-isomaltase (SI), maltase-glucoamylase (MGAM), andlactase. These enzymes, along with trehalase (a minor player), arecontinuously produced by healthy luminal enterocytes that constitute theapical mucosal surface, also referred to as the brush border because ofthe exposed velveteen-appearing microvilli. Released monosaccharides,mostly free glucose, enter the absorbing enterocyte at the apicalsurface by specific transport mechanisms (“carriers”) and aredistributed to the body and inhibit hepatic gluconeogenesis. It is wellknown that mucosal enzyme activity is frequently reduced in states ofmalnutrition (Mehra et al., 1994) and mucosal injury. Except for lactaseand sucrase, this mucosal-level enzyme insufficiency that completesstarch and fructan hydrolysis (maltase and isomaltase) is a largelyunaddressed clinical and therapeutic concern.

The complete digestion of starch is thought to be predominantly relianton the proper expression and function of the sucrase-isomaltase (SI)enzyme complex (Auricchio et al., 1963). The SI is a complex composed oftwo [alpha]-glucosidase units, sucrase, and isomaltase (Conklin et al.,1975). The isomaltase component demonstrates considerable[alpha]-glycosidic activity on starch-derived glucose oligomers, and theSI complex together contributes to 60 to 80 percent of total intestinalmaltase activity (Gericke et al., 2016). The isomaltase component of theSI complex hydrolyzes [alpha]-1,6 linkages of the [alpha]-limit dextrinsafter primary luminal pancreatic amylase has reduced the ingested starchto soluble subunits (Galand, 1989). Generally, mucosalmaltase-glucoamylase (MGAM) is relatively restricted to hydrolyze alpha1-4 bonds of soluble oligosaccharides and together with the SI componentworks to hydrolyze alpha 1-6 bonds and complete starch digestion toglucose with some cross activity between enzymes (Diaz-Sotomayor et al.,2013). MGAM is noted to have a significant amount of compositional aminoacid sequence homology that appears responsible for some evolutionaryredundancy for survival. Congenital mucosal enzyme deficiency syndromeshave been reported due to primary gene mutations (Geng et al., 2014) andsecondary transport errors (Jacob et al., 2000).

In western societies, where dairy products remain part of the habitualdiet, adult-type hypolactasia presents a challenge to those unwilling torefrain from non-fermented/cured products containing lactose. Symptomsof gastrointestinal distress, identical to those described above, may bemitigated by a nutritional enzyme supplement, called LACTAID® thatcontains the microbial-produced enzyme lactase-phlorizin hydrolase. Theoral supplement is typically a gel-capsule containing microbial producedlactase (−9000 LU/capsule) and is a model approach to alleviatingsymptoms of other digestive enzyme insufficiency syndromes. Twoubiquitous, GRAS-listed, filamentous fungi, Aspergillus oryzae andAspergillus niger, or recombinant yeast organisms are used tocommercially produce lactase for human dietary supplementation withoutobjection from the US Food & Drug Administration.

In a manner similar; a yeast-derived enzyme product, sacrosidase, hasbeen put forth to aid in the digestion of sucrose (Puntis and Zamvar,2015), but it has virtually no starch or fructan hydrolyzing activity(Kasperowicz et al., 2012). The utility of sacrosidase is adjunctivesince it is often easier to restrict dietary sucrose, but a similarapproach cannot be applied to dietary starch maldigestion becausedietary starch is a human staple. Recombinant human SI and MGAM are notwidely available, and stability has not yet been determined, so otherexogenous alternatives are needed clinically to aid faulty starchdigestion when symptomatic problems occur.

Enteritis is a dysfunctional state in which destruction of thegastrointestinal epithelial mucosa (small intestine) occurs with theresultant loss of digestive enzyme expression with severe symptoms, andthis typically occurs as a consequence of enteric infection, such asrotavirus (Langman and Rowland, 1990). The mucosal cells of the smallbowel are highly metabolic, are known to turn over rapidly and areparticularly vulnerable to the toxic effects of viruses, parasites, andto some medications used to treat infections and malignancies. Mucosalsurface enterocytes are not the only primary digestive agents but are aprimary line of defense against gastrointestinal infections, such asecondary cryptosporidiosis or giardiasis. In a manner of speaking,enterocytes take the first hit. In addition to various infections,interruption of mucosal digestive processes by toxins, andprotein-calorie malnutrition can further impact normal digestiveprocesses and predispose to protracted disease (Custodio et al., 2000;Hlaysa et al., 2016). For example, HIV infection is a major predisposingfactor leading to secondary infections and resultant wasting andsecondary intestinal disaccharidase deficiency (Taylor et al., 2000).Enzyme deficiency may idiopathically occur in the absence of visiblemucosa injury. Enzyme inhibition by binding (e.g., acarbose) (Singla etal., 2016), and substrate mimicry (Bompard-Giolles et al., 1996), alsointerferes with starch hydrolysis and may cause significant morbiditydue to food maldigestion.

The use of combination radiochemotherapy for the management of rectalcancer, pancreatic adenocarcinoma (Wang-Gillam et al., 2013), and otherabdominal tumors (Mundt et al., 1999) is known to produce predictableadverse side effects such as mucositis-associated diarrhea. Enterocytedamage appears to worsen in severity with the type of oncotherapy (Reiset al., 2015). The high rate of gastrointestinal mucosal cellularreplication affords the mucosa a particular-susceptibility to this typeof cytotoxicity. The condition is similar, but often greater severitythan infectious enteritis, and reflects another unmet clinical need toaid or replace digestive enzyme activities at the mucosal surface level.Like infectious enteritis, mucositis is characterized by reducedproliferation of epithelial cells in the intestinal crypts and villousatrophy, which results in a loss of absorptive capacities (Keefe et al.,2000). Recovery is variable and appears dependent upon multiple factorsinherent to the primary disease in addition to inadequate nutrientdigestion and absorption.

Mucositis may be very severe in patients who receive aggressivemyeloablative chemotherapy and for some aggressive gastrointestinal andpelvic malignancies. It is a significant problem that affectsapproximately one-third of patients treated with adjunctive pelvicradiochemotherapy for rectal cancer which often prompts a modificationin primary treatment (Roh et al., 2009). Mucositis predisposes tosupra-infections (Honda et al., 2010). During the time of malignantproliferation or infectious insult, glucose and other nutrients neededfor cell expansion are derived, in-part, from accelerated apoptosis ofhealthy cells and breakdown of lean body mass to capture recyclednutrients (cannibalistic wasting secondary to inflammatory processes)(Fearon et al., 2011). In summary, malignancy inherently creates anenergy deficit (cachexia), immune suppression and mucositis impairsabsorption of nutrients including glucose derived from dextrin andoligosaccharides to compound the scene. In health, the small intestinalglucose transporter is efficient (Wright et al., 2011) and in disease isamong the first apical surface turnover proteins to be expressed inrecovery (Klish et al., 1980) and appears to precede expression ofmucosal enzymes and deserving of exploitation. It appears that specificadjunctive digestive enzyme therapy that releases glucose fromstarch-derived oligosaccharides could be beneficial toward restoringpositive energy balance, and demand for this type of therapy would begreat.

The loss of small bowel enterocytes decreases the availability ofmucosal bound and mucosal secreted digestive enzymes and results inmaldigestion of food substrates that ultimately leads to excessivecolonic fermentation of unabsorbed nutrients, including startch, andresultant manifested symptoms include bloating, pain, fatigue, anddiarrhea (Stringer et al., 2007). These distal events may be associatedwith loss of structural collagen and macroscopic ulcerations withbleeding and loss of iron stores. Furthermore, the onset of diarrhea canfurther alter dietary patterns, lead to dehydration, electrolyteimbalance, and lead to perirectal skin breakdown, local pain, andcellulitis (Peterson et al., 2011). As such, enteritis and mucositis aresignificant clinical problems because the effects predispose the patientto comorbidities including malnutrition, anemia and additionalopportunistic infection (Andreyev et al., 2014). With thesecomorbidities comes further net loss of mucosal enzyme expression tocompound the disease process.

The features of enteritis are similar to those of irritable bowelsyndrome, parasitic enteritis, small bowel bacterial overgrowth, orrunner's diarrhea (de Oliveira et al., 2017) and associated with lessmorbidity. Symptoms may include abdominal pain, bloating, changes instool patterns and variable central nervous system disturbance (e.g.headaches), and the symptoms may be incapacitating (Marteau and Flourie,2001). In lieu of endoscopy, substrate-specific, tracer breath testtechnology may be used to identify patients that have lost enzymaticdigestive functions for starch-derived oligosaccharides but these testsare not widely available, but have been optimized for use in clinicalfield studies (Opekun et al., 2014).

Proximal maldigestion leads to excessive distal colonic fermentation andproduction of fatty acids that, when absorbed, may indirectly contributeto anorexia to complicate a clinical condition (Petersen and Forsmark,2002) and colitis. Proximal maldigestion can also lead to adversechanges in the distal colonic microbiota (Nielsen et al., 2016). Theloss of mucosal integrity also contributes to inflammatory responsesthat further impede enterocyte recovery, including loss of theexpression of nutrient transporter expression with concomitantnon-passage of free luminal D-glucose (Cardani et al., 2014). EnterocyteNa—K-ATPase activity that drives co-transporters become diminished (Sahaet al., 2015) and may contribute to increased vulnerability tolipopolysaccharide-induced mucosal injury and exacerbation ofmalabsorptive conditions. As such, it stands to reason that theavailability of free D-glucose to some of the spared enterocytes, wherethe normal sodium glucose transporter 1 (SGLT-1) persists, would becritical to restoring mucosal health. Amelioration of the malabsorptivecondition could limit further injury. Simply stated, local cells in theintestines need energy from free glucose to survive and heal, and atargeted digestive aid for starch-derived oligosaccharides should bevery useful in gut rehabilitation. As such, increasing starchdigestibility appears to be a sound approach to therapeuticallyincreasing mucosal glucose availability when endogenous mucosal enzymesare insufficient and when glucose transporters remain intact or arefirst to recover.

Treatment of enteritis, a primary cause of carbohydrate intolerance,mainly consists of controlling symptoms by inhibiting motility andsecretions with highly variable results (Schiller, 2017). Agents thatsuppress intestinal motility (e.g., loperimide) and octreotide are themainstay-treatment that inhibits secretions, assisting with thelimitation of volume depletion, but these agents do so without improvingdigestive capacities. At present, there is no widely accepted approachto aid digestion of dietary carbohydrate oligomers (oligosaccharidesdextrins, starches). In advanced settings, elemental feeding andintravenous feeding may be implemented at great cost and iatrogenicrisk. Yeast derived sacrosidase (aka: invertase) has been used to assistthose with congenital sucrase deficiencies and other mucosalinflammatory conditions and is a parallel model (different substrate anddifferent enzyme) to approach the current starch maldigestion problem(Cohen, 2016; Puntis and Zamvar, 2015). Numerous, low-dose supplementshave been marketed without specific indications, activity, or purity andefficacy remains to be proven. Effective facilitation of compromisedcarbohydrate digestion would be expected to limit aberrant colonicfermentation and its associated osmotic and inflammatory effects, andtherefore decrease diarrhea and related mucositis symptoms. Furthermore,and conversely, enhancement of digestion of nutritionally sparsestarchy-grains, such as sorghum, quinoa, spelt, and amaranth couldenhance caloric yield among the undernourished populations living inarid conditions.

This application and disclosure encompasses the embodiment thatingestion of non-toxic fungal-derived enzymes (e.g., amyloglucosidase,AMG) could significantly aid in the hydrolysis of dietary starches whendelivered and dosed appropriately (CAS Number: 9032-08-0).Administration of AMG should be helpful as a nutraceutical or medicalfood to release glucose in the stomach or small intestine, with orwithout the addition of invertase (sacrosidase) (CAS Number: 9001-57-4)(del Castillo Agudo and Gozalbo, 1994). Numerous dietary supplements aremarketed that contain small amounts of AMG to aid starch digestion.There is one, small double-blind clinical trial, from 1971 by Karina etal., that reported symptom improvement with the use of a crude,fungal-derived enzyme concentrate that contained AMG (Karani et al.,1971). As such, the concept of specifically using AMG to aid thecomplete digestion of starch has not been thoroughly pursued.Furthermore, the concept has been suggested for use in animals thatingest crude plant cell wall grains (B-glucans in barley and oats andarabinoxylans (pentosans) in rye and wheat) to improve digestibilityand, to that end, weight gain (Cambell G L and Bedford M R, 1992), butnot specifically nor conclusively for dietary starch contained withinthe kernels.

Invertase is biosynthesized by many microorganisms including yeaststrains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis(Para et al., 1980) and two forms of invertase exist, with and withoutcarbohydrate moieties. It is loosely understood that sucrose-hydrolyzingenzymes, derived from plants, are called invertase because a sucrosesubstrate solution polarizes light in a positive direction, but afterhydrolysis to fructose and glucose, the net light rotation is −20degrees. The external form of invertase is predominant; it is aglycoprotein containing 50% mannan and 2-3% glucosamine with a molecularweight of ˜260 kD. The intracellular invertase has a molecular weight of135 kD and is relatively void of carbohydrates. It is unlikely that theaddition of invertase could offer any clinically significant advantageswhere starch maldigestion is thought to be the main problem. It isunclear if invertase that hydrolyzes sucrose(α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside, EC 200-334-9), could, incombination, offer additional benefit as sucrose is also pervasive inthe diet of western populations, but whereas sucrose is relatively voidfrom the diet elsewhere (Wittekind and Walton, 2014). Invertase, fromsome sub-species of Saccharomyces, may have some inherent glycosidicactivity for polysaccharides because it has been shown to hydrolyzeinulin (Latorre-Garcia et al., 2005; Yuan and Wang, 2013), but itsutility to aid in starch or dextrin hydrolysis is minimal or null.Sacrosidase activity is commonly measured as Sumner units, and this isthe activity of the enzyme which, under the conditions of the assay,will convert 1 mg of sucrose to glucose and fructose in 5 minutes.

Amyloglucosidase (AMG) as a potential nutraceutic for maldigestion andmalnutrition

AMG is derived from non-pathogenic strains of Aspergillus species andSaccharomycopsis and, like MGAM, is inhibited by acarbose (Amirul etal., 1996; Sauer et al, 2000). AMG is expressed in vivo with a varietyof other noxious organic compounds and recombinant, pure forms are notyet known to exist. AMG has a reported specific glycosidic activity thatapproximates 330 amyloglucosidase (glucoamylase) units (AGU) permilligram protein (Natarajan and Sierks, 1996), is robust with regard totemperature and pH activity and can be selectively precipitated fromcrude culture filtrate by alginate (affinity precipitation) (Teotia etal., 2001; Mahajan et al., 1983) [Foods Chemical Codex (FCC-USP)activity unit of glucoamylase activity has also been defined as theamount of glucoamylase that will liberate 0.1 μmol/min of p-nitrophenolfrom the PNPG Solution at pH 4.5 and 50° C. on a casein substrate] whichrepresents hydrolysis of one maltose equivalent.

AMG hydrolyses dietary starch, oligosaccharides, dextrins,alpha-limit-dextrins, and alpha-cylodextrins. Numerous nutritionalsupplements have been marketed that contain trivial amounts of crudeAMG, but clinical efficacy, specific to AMG, has not been previouslyclaimed. AMG is a potent inverting exo-acting hydrolase-releasingglucose from the non-reducing ends of oligosaccharides which is used inminuscule amounts as a component of nutritional supplements and in thefood industry to modify starches in vitro (Guzman-Maldonado andParedes-Lopez, 1995). Relative rates of hydrolysis for AMG at 37° C. andpH 4.8 for some representative oligosaccharides are: (maltose: 300:maltotriose: 360; maltotetrose: 770; maltopentoses: 1000;iso-maltopentose: 23; and 6-α-maltosylglucose: 260. AMG easilyhydrolyses dietary starch, but the rates are highly variable dependingupon the agricultural grain source. AMG has been shown to hydrolysealpha-cyclodextrins, a type of dietary emulsifier may causegastrointestinal distress and malabsorptive/fermentative diarrhea whenexcessively taken in (Lina et al., 2004), but the AMG-hydrolysis ratesare highly variable depending upon ring size (Rather et al., 2015, Chenet al., 2017) and luminal conditions. AMG will not cleave Glu α1-2-Frubonds (as in sucrose) or glucan bonds in chemically-modified starch(e.g., with >1% of glucose product of polysaccharide hydrolysis isD-glucose). It is distinguished by releasing alpha-glucose fromsubstrates having alpha-glycosidic linkages (Goncalvez et al., 1998) andis used in the final step of food analysis to determine soluble dietaryfiber content from insoluble content (Nisha and Satyanarayana, 2016).The free enzyme is active in aqueous media and lipid vesicles (Li etal., 2007). Three-dimensional structures have been determined for freeand inhibitor-complexed glucoamylases. The catalytic domain folds are atwisted (alpha/alpha) (6)-barrel with a central funnel-shaped activesite, while the starch-binding domain folds as an antiparallelbeta-barrel and has two binding sites for starch (Sauer et al., 2000).

Allergy to inhaled AMG and to β-xylosidase (often concomitantlyexpressed) has rarely been reported (Quirce et al., 2002; Sander et al.,1998) but is highly unlikely to be of any significant concern sinceingested enzyme that will ultimately be digested itself as a proteinfood substrate.

The presence of mycotoxins sometimes are a consideration whenconsidering the use of mold (fungal cultures) for production ofbiologics for use in humans or animals. Of concern herein is theco-expression of several low molecular weight aflatoxins and metabolitesthat rarely may be expressed with AMG by certain Aspergillus species(Wilson et al., 2002) The FDA places strict limits on the amount ofseveral aflatoxins may be permitted to be present in foodstuffs(Mazumder and Sasmal, 2001). In contrast, the trichothecenes (e.g.,cyclic sesquiterpenoids), deoxynivalenol (DON) is one of the most commonnuisance contaminants of various foodstuffs and has been reported to beproduced by Fusarium genus and some species of Aspergillus (Milicevic etal., 2010; Pieters et al., 2002). Specifically, it was discovered thatDON was present in food grade AMG and is herein first reported with thispresent application and disclosure. AMG was originally intended forsmall, homeopathic dosages and it was determined to be problematic whendosages were efficaciously increased to increase dietary starchdigestion in humans. As the dose of AMG was increased, so too was theconcentration of DON concomitantly increased. DON is potent, lowmolecular weight mycotoxin that has been reported may lead toself-limiting vomiting, diarrhea, and other symptoms that affect thecholinergic receptors and is not known to cause serious or lastingillness (Pestka and Smolinski, 2005). Trichothecenes, of which DON is aconstituent class member, are reactive species and removal from thefinal composition will extend the stability and shelf-life of the finalcomposition (AMG). Some trichothecenes can be tested for and are removedby decanting lipid layer when crude AMG is put in aqueous solution(Koch, 2004; Fernandez et al., 1994; Papageorgiou et al., 2018) or byabsolute alcohol precipitation of AMG and extraction followed by assay.The universal limit for DON ingested is 1 μg/kg body weight/day for DONand its related metabolites and this represents a significant limitingfactor for expanded clinical use. It appears that DON may be a markerfor other trichothecenes or DON-derivatives that may or may not beclinically toxic, but not readily detected and, in a similar way, mostor all toxins should be removed prior to clinical use of AMG.

Ochratoxin A (MW 403.8D) is of modest concern since it presence isunlikely since it is not known to be associated with AMG-producingAspergillus niger (only A. carbonarius which is not the subject of thispresent disclosure) (Abarca et al, 1994; Trenk and Chu, 1971). It ismentioned herein because it is potentially carcinogenic to human, is aneurotoxin, and is potentially nephrotoxic fat soluble ochratoxin A maybe present in low concentrations in AMG if other species, includingPenicillium verrucosum, P. nordicum, were aberrantly introduced to thebioreactors; pointing to the need to source only quality raw materials(Stoll et al, 2013) and purify the composition. FDA limits theappearance concentration of Ochratoxin A to less than 50 ppb, and AMGderived from pure cultures of A. niger universally meets thisrequirement (Malir et al, 2001). Ochratoxin A is also highly soluble inethanol (10-50 mg/mL). Commercial, ISO/IEC 17025:2005 certifiedlaboratories are available to routinely test for some mycotoxins incrude or finished products.

The present application and disclosure satisfies a long-felt need in theart of compositions for enhancing starch digestion, preventingmaldigestion, relieving the symptoms of maldigestion, and increasingcaloric yield and includes methods for preparing the compositions.

BRIEF SUMMARY

The present disclosure is directed to methods and compositions relatedto the toxin-free enhancement of starch digestion and caloric yield formammals, including humans, dogs, cats, cows, horses, goats, sheep, pigs,and so forth. Methods of producing the compositions are also encompassedin the disclosure. In particular embodiments, the methods andcompositions are related to the production and/or use of particularformulations of amyloglucosidase (AMG). In specific embodiments, the AMGis particularly concentrated and formulated to lack one or more toxins.The one or more toxins may be intentionally excluded from the AMGformulations. In many cases, the formulations are produced with theintent of making the AMG formulations to have fewer or no toxinscompared to crude or unprocessed formulations of AMG. In specificembodiments, the formulations are toxin-free or have substantiallyreduced levels of one or more toxins compared to other formulations and,as such, the concentrated, toxin-free compositions confer clinicalutility.

Production methods for the AMG formulations are able to refine,concentrate, and make toxin-free amyloglucosidase, in specificembodiments. In some cases, the AMG formulation is present in aparticular form, such as a capsule, for example. In certain cases, theAMG formulation comprises an amount of AMG that is other than an amountof known formulations. In specific cases the AMG amount is able to behigher than known formulations because the AMG lacks one or more toxinsthat standard AMG formulations have.

In specific embodiments, methods of production allow for production ofAMG formulations lacking deoxynivalenol “vomit toxin” that is present inknown formulations, including formulations having the crude enzyme. Themethods of production may include one or more particular ethanol washingsteps that eliminate the deoxynivalenol and its related metabolites froma crude enzyme preparation, for example. Such washing steps may reducethe amount of deoxynivalenol at least 10-fold or more with each absoluteethanol wash/extraction step, in at least some cases.

In particular embodiments, AMG formulations are used foramyloglucosidase supplementation in congenital sucrase isomaltase, smallbowel mucositis, functional bowel disorders (including irritable bowelsyndrome and functional dyspepsia, for example), functional bowelsyndrome, functional gastroduodenal disorders, and protein calorie(energy) malnutrition, for example. Use of the formulations allowsimproved complex carbohydrate digestion that prevents at least excessivecolonic fermentation, decrease inflammation and osmotic diarrhea uponincreasing caloric absorption. The methods and compositions of thedisclosure assist the body in normal digestion and assimilation of foodstarch.

Embodiments of the disclosure include methods of optimizing starchdigestion in an individual, comprising the step of providing to theindividual an effective amount of a composition comprisingamyloglucosidase. The composition may comprise amyloglucosidase in adosage that is equal to or greater than 5,000 unit releases of one gramof glucose per hour (AGU), in at least some cases. The composition maybe provided to the individual orally and it may occur daily, weekly,monthly, or yearly, or more than once daily. The individual may or maynot be healthy or malnourished but may be in need of treatment orprevention of congenital sucrase isomaltase syndrome, functional boweldisorders, functional bowel syndrome, functional gastroduodenaldisorders, small bowel bacterial overgrowth, radiochemotherapy-inducedmucositis and/or short-gut syndrome. The individual may be an infant,child, adolescent, teenager, or adult.

The AMG composition may be formulated in any appropriate manner but inspecific embodiments is formulated in a comestible or beverage. The AMGmay be a food ingredient, medical food, dietary supplement,nutraceutical, and/or drug preparation. The AMG composition may be inthe form of a solid, liquid, or gel. It may be a capsule, tablet, pill,film, lozenge, powder, or combination thereof.

In some embodiments there are methods for producing a purifiedamyloglucosidase composition comprising extracting a crudeamyloglucosidase composition in the presence of an immiscible solvent,and separating a purified amyloglucosidase composition from theimmiscible solvent. The immiscible solvent may be absolute ethanol,acetone, dimethyl sulfoxide, methanol, chloroform, diethyl ether, or acombination thereof. In particular cases the solvent from which thepurified amyloglucosidase composition is separated comprises one or moredetectable or non-detectable toxins. The crude amyloglucosidasecomposition may be in the form of a solid. In particular embodiments,the step of extracting comprises vortexing the crude amylglucosidasecomposition and the immiscible solvent. Isolating the retained, purifiedamyloglucosidase composition may comprise spinning down theamyloglucosidase composition, such as in a centrifuge. The method mayinclude repeating the extracting and isolating steps one or more times,including two or more times. The method may further comprise dissolvingthe retained amyloglucosidase composition in water to form a solution,and filtering the solution.

The crude amyloglucosidase may be supplied in the form of a liquid or asolid. When the crude amyloglucosidase is in the form of a liquid, thecrude liquid amyloglucosidase may be admixed with an inert excipient andfollowed by purification processing. When the crude amyloglucosidase isin the form of a solid, it may have been previously admixed with anorganic excipient, such as starch or maltose or a relatively inertexcipient(s) such as a calcium salt. In some embodiments, there is amethod for optimizing starch digestion in an individual, comprising thestep of providing to the individual an effective amount of anamyloglucosidase composition produced by a method encompassed by thedisclosure.

In certain embodiments, there are methods of optimizing starch digestionin an individual, comprising the step of providing to the individual(such as orally) an effective amount of a composition comprisingtoxin-free amyloglucosidase (AMG), wherein the effective amount is at adosage range 5,000-20,000 AGU, although in some cases the effectiveamount is at a dosage range lower than 10,000 AGU or higher than 20,000AGU. The AMG may be released at 5,000-20,000; 5,000-15,000;5,000-12,000; 5,000-10,000; 5,000-9,000; 5,000-8,000; 5,000-7,000;5,000-6,000; 6,000-20,000; 6,000-15,000; 6,000-12,000; 6,000-10,000;6,000-8,000; 7,000-20,000; 7,000-15,000; 7,000-12,000; 7,000-10,000;8,000-20,000; 8,000-15,000; 8,000-12,000; 8,000-10,000; 6,000-10,000;6,000-9,000; 6,000-8,000; 6,000-7,000; 7,000-10,000; 7,000-9,000;7,000-8,000; 8,000-10,000; 8,000-9,000; 9,000-10,000; 10,000-20,000;10,000-19,000; 10,000-18,000; 10,000-17,000; 10,000-16,000;10,000-15,000; 10,000-14,000; 10,000-13,000; 10,000-12,000;10,000-11,000; 11,000-20,000; 11,000-19,000; 11,000-18,000;11,000-17,000; 11,000-16,000; 11,000-15,000; 11,000-14,000;11,000-13,000; 11,000-12,000; 12,000-20,000; 12,000-19,000;12,000-18,000; 12,000-17,000; 12,000-16,000; 12,000-15,000;12,000-14,000; 12,000-13,000; 13,000-20,000; 13,000-19,000;13,000-18,000; 13,000-17,000; 13,000-16,000; 13,000-15,000;13,000-14,000; 14,000-20,000; 14,000-19,000; 14,000-18,000;14,000-17,000; 14,000-16,000; 14,000-15,000; 15,000-20,000;15,000-19,000; 15,000-18,000; 15,000-17,000; 15,000-16,000;16,000-20,000; 16,000-19,000; 16,000-18,000; 16,000-17,000;17,000-20,000; 17,000-19,000; 17,000-18,000; 18,000-20,000;18,000-19,000; or 19,000-20,000 AGU. The individual may be in need oftreatment or prevention of congenital sucrase isomaltase syndrome,functional bowel disorders, functional bowel syndrome, functionalgastroduodenal disorders, small bowel bacterial overgrowth,radiochemotherapy-induced mucositis and/or short-gut syndrome. In somecases the providing step occurs daily, more than daily, weekly, monthly,or yearly. The individual may be healthy or malnourished, and theindividual may be an infant, child, adolescent, teenager, or adult. Inany case, the composition may be formulated in a comestible or beverage,including in a food supplement. The AMG composition may be in the formof a solid, liquid, gel, or mist, including as a capsule, tablet, pill,film, lozenge, powder, or combination thereof.

In some embodiments, there is a toxin-free AMG composition, or an AMGcomposition comprising substantially reduced levels of toxin withreference to an AMG composition that has not been subjected to at leastone extraction/purification step. Toxin-free AMG compositions producedby methods described herein are encompassed by the disclosure. Thecomposition may be present in a capsule, tablet, pill, film, lozenge,powder, or combination thereof. The amyloglucosidase may be in a dosagethat is equal to or greater than 10,000 unit releases of one gram ofglucose per minute.

The foregoing has outlined rather broadly the features and technicaladvantages of the present disclosure in order that the detaileddescription that follows may be better understood. Additional featuresand advantages will be described hereinafter which form the subject ofthe claims herein. It should be appreciated by those skilled in the artthat the conception and specific embodiments disclosed may be readilyutilized as a basis for modifying or designing other structures forcarrying out the same purposes of the present designs. It should also berealized by those skilled in the art that such equivalent constructionsdo not depart from the spirit and scope as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe designs disclosed herein, both as to the organization and method ofoperation, together with further objects and advantages will be betterunderstood from the following description when considered in connectionwith the accompanying figures. It is to be expressly understood,however, that each of the figures is provided for the purpose ofillustration and description only and is not intended as a definition ofthe limits of the present disclosure.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the disclosure may apply to any otherembodiment of the invention. Furthermore, any composition of thedisclosure may be used in any method of the invention, and any method ofthe disclosure may be used to produce or to utilize any composition ofthe invention. Aspects of an embodiment set forth herein are alsoembodiments that may be implemented in the context of embodimentsdiscussed elsewhere in the application, such as in the Summary ofInvention, Detailed Description, Claims, and description of FigureLegends.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure, referenceis now made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 : The proto-type AMG nutritional food supplement prepared underclean conditions for further in-vitro testing. Each capsule as oneexample may be 8 mm×24 mm in dimension or larger (10 mm×28) mm orsmaller (5 mm×15 mm) and contains approximately 500 mg. powder contentor more (approximately 750 mg.) or less (approximately 250 mg.), most ofwhich is neutral excipient used to deliver dry enzyme duringpreparation. Each capsule has approximately 4000 AGU enzymatic activityon maize and 8000 AGU enzymatic activity, or more, on sorghum starch ormaltodextrin or dextrin from corn.

FIG. 2A: SDS-PAGE Results for Amyloglucosidase. The SDS PAGE results forAMG samples 26452, 26454, 26456, 26457, 26459 and 26461, along withSigma references for A. niger A7420 and A. rhizopus A9228. The expectedresults include glycoprotein bands at ˜90 kD and a band at ˜70 kd. Allsamples are consistent for AMG, except most dry samples containadditional protein bands as ˜60 kD that that probably represent enzymefragment. This is faintly seen in the Sigma A4720 reference as well.Invertase control is distinguished as a distinct entity.

FIG. 2B: SDS-PAGE Results for Amyloglucosidase. The SDS PAGE results forAMG samples Ultrabiologic's Nutritek AMG samples (26424B), along withSigma references for A. niger A7420 (26425). The intensity for the bandsis remarkable and consistent with the 53% crude protein content forsample 26424B.

Reduced Gel: Lane 1—Molecular Weight Markers; Lane 2—Sample 700126425;Lane 3—Sample Filtered AMG (700126424A) before processing; Lane 4—SampleFiltered AMG (700126424B) before processing; Lane 5—Sample Filtered AMG(700126424A) after processing; Lane 6—Sample Filtered AMG (700126424B)after processing

Non-Reduced Gel: Lane 7—Molecular Weight Marker; Lane 8—Sample 700126425NR; Lane 9—Sample Filtered AMG (700126424A) before processing NR; Lane10—Sample Filtered AMG (700126424B) before processing NR; Lane 11—SampleFiltered AMG (700126424A) after processing NR; Lane 12—Sample FilteredAMG (700126424B) after processing NR

FIG. 3 : Thin-layer chromatography results for amyloglucosidase samples.The TLC results for AMG samples 26452, 26454, 26456, 26457, 26459,26461, 26461-dialyzed along with Sigma references for A. niger A7420(internal #301N). All commercial AMG samples demonstrate LMW bandsconsistent with typical impurities and flavonoids (i.e., isoflavoneaglycones) and/or excipients, except for AMG 26461 and Sigma controlsamples. Sigma sample 26461 was provided as a crude liquid (syrup) andprobably was not adulterated. Samples BIOCAT 26556 and NEC 26457 showaberrant low bands suggestive of mycotoxin (deoxynivalenol, aka: VOMITTOXIN) or other contaminants that might signal the presence ofdeoxynivalenol. The importance of dialysis, filtration or some othertechnique (gel filtration) to remove residual biological substances isconsidered. (n.b., the presence of isoflavone aglycones, such asdaidzein, genistein and glycitein are suspected because of typicalfementation aroma.)

FIG. 4 : Repeat thin-layer chromatography results for Ultrabiologic[Nutritek®] amyloglucosidase samples 26437C (crude) in solution and26437R (refined) in citrate buffer (pH 4.5). Refined samples (15 mg/mL)were filtered and concentrated as per SOP. HPLC grade AMG (Sigma A7420TLC is shown for comparison and is void of extraneous components. Theimportance of dialysis or come other purification technique (gelfiltration) to remove residual biological substances is considered.

FIG. 5 : A glucoamylase enzyme activity assay using a variety ofsubstrates.

FIG. 6 : A BioCat AMG dialyzed enzyme activity assay with a variety ofsubstrates.

FIG. 7 : A NEC AMG dialyzed enzyme activity assay using a variety ofsubstrates.

FIG. 8 : A Creative Enzymes AMG dialyzed enzyme activity assay using avariety of substrates.

FIG. 9 : Production comparison of processed Ultrabiologic® material whenhydrolyzing sorghum compared to AMG capsule when hydrolyzing sorghum.

FIG. 10 : Production comparison of processed Ultrabiologic® materialwhen hydrolyzing rice compared to AMG capsule when hydrolyzing rice.

FIG. 11 : Production comparison of processed Ultrabiologic® materialwhen hydrolyzing wheat compared to AMG capsule when hydrolyzing wheat.

FIG. 12 : Production comparison of processed Ultrabiologic® materialwhen hydrolyzing maize compared to AMG capsule when hydrolyzing maize.

FIG. 13 : Production comparison of processed Ultrabiologic® materialwhen hydrolyzing millet compared to AMG capsule when hydrolyzing millet.

FIG. 14 : Mean Glucose Concentration with AMG and Maltose.

FIG. 15 : 3-hour ¹³C-starch tracer labeled rice pudding breath testresponse to amyloglucosidase meal supplementation (20K AGU) in 4symptomatic subjects with congenital sucrase-isomaltase deficiencysyndrome (3p25.2-26). Each data point was adjusted for inherent CO2production rate using Scofield equations.

FIG. 16 : The change (delta) in ¹³C-starch breath-test response frombaseline, measured in humans at 120 minutes elapsed time, to one oraldosage of purified amyloglucosidase. Column left shows a robust responseto 20,000 AGU amyloglucosidase in a patient with CSID, and indicatesthat a dose-response relationship exists, and column right shows arobust response to 20,000 AGU in a patient with mixed-form irritablebowel syndrome (IBS-M). The non-response (left column 4000 AGU)indicates that the response was too small to be detected or that thereleased glucose was assimilated into body stores and that there was noexcessive glucose available for oxidation and breath test detection at120 minutes ET.

FIG. 17 : The change (delta) in blood glucose concentration responsefrom baseline, measured in humans at 45 minutes elapsed time, to oneoral dosage of purified amyloglucosidase. Column-pair left shows arobust response to 4,000 AGU amyloglucosidase in a patient with CSID;and column-pair right shows a robust response to 4,000 AGUamyloglucosidase in a patient with mixed-form irritable bowel syndrome(IBS-M).

DETAILED DESCRIPTION

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the invention. It is contemplated thatany method or composition described herein can be implemented withrespect to any other method or composition described herein.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used herein, the terms “or” and “and/or” are utilized to describemultiple components in combination or exclusive of one another. Forexample, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone,“x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” Itis specifically contemplated that x, y, or z may be specificallyexcluded from an embodiment.

Throughout this application, the term “about” is used according to itsplain and ordinary meaning in the area of cell and molecular biology toindicate that a value includes the standard deviation of error for thecomposition or method being employed to determine the value.

The term “comprising,” which is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. The phrase“consisting of” excludes any element, step, or ingredient not specified.The phrase “consisting essentially of” limits the scope of describedsubject matter to the specified materials or steps and those that do notmaterially affect its basic and novel characteristics. It iscontemplated that embodiments described in the context of the term“comprising” may also be implemented in the context of the term“consisting of” or “consisting essentially of.”

The term “toxin-free” as used herein refers to lacking one or moretoxins in comparison to a standard formulation. In specific cases,toxin-free refers to lacking deoxynivalenol (vomit toxin), includinglacking detectable levels of vomit toxin. In alternative cases, aformulation may have detectable level of vomit toxin but the level issubstantially reduced following production methods of the disclosure. Incertain embodiments, the level is decreased to a concentration belowmaximum acceptable levels deemed by regulatory authorities. In someembodiments, a particular way to reduce the level of deoxynivalenol(vomit toxin) is by serial absolute organic solvent washes (absoluteethanol extractions) until low or undetectable levels of deoxynivalenolare achieved, as measured by gas chromatographic mass spectrometry or bythin-layer chromatography or by use of commercial ELISA assay, such asDON-V® (VICAM, a Waters Business, Milford, Mass. 01757. Thesubstantially reduced level of vomit toxin may be reduced in aformulation compared to the level in a starting or crude formulation,such as reduced by a level that is 10, 20, 30, 40, 50, 60, 70, 80, 90,91, 92, 92, 94, 95, 96, 97, 98, or 99% lower than a starting or crudeformulation or by direct testing using ELISA (less than 0.05ppm-per-gram of purified AMG). In particular embodiments, there is lessthan 0.05 parts per million (ppm) per gram of refined (washed) AMG. Insome embodiments, there is less than 0.5, 0.4, 0.3, 0.2, 0.1, 0.09,0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 ppm per gram ofrefined (washed) AMG.

I. General Embodiments

Survival in healthy or diseased states depends on an adequate andcontinuous supply of energy in the form of glucose delivered to allcells in the body. Approximately half of the body glucose needed forenergy needs is derived from dietary starches, with the balance beingderived from a variety of sources including dietary and stored fat,protein stuffs, and glycogen stores. Malnourished children, living inremote parts of sub-Saharan Africa, have limited energy stores and areunable to fully assimilate the relatively dilute nutritional content ofsome dietary staple grains, including millet, sorghum, maize and cassavaand due to limited digestibility, especially during states of chronicillness.

For example, in Tanzania, poverty and food insecurity are the maindrivers of chronic under-nutrition and is indirectly responsible formore than 130 child deaths every day. There are 8 million Tanzanianchildren aged less than five-years old, and 42% have stunted growth and16% are considered underweight and recurrent diarrhea and failure toassimilate dietary calories is the primary putative factor. Most workhas focused on the means to increase protein derived from grains, butthe need to increase energy yield from dietary starch (polysaccharides)is also important. In addition to caloric intake, micronutrientdeficiencies are widespread and parallels caloric intake, with more thanhalf of malnourished children, aged less than five years, are alsoconsidered anemic. Childhood malnutrition is most prevalent (50 percentor higher) in the regions of Dodoma, Iringa, Mbeya, Njombe, Rukwa, andLindi, so a method to increase caloric assimilation from nutritionallysparse grains is needed.

This disclosure addresses the embodiment that starch digestion andcaloric yield can be enhanced by optimizing luminal carbohydratedigestion with an orally administered supplemental enzyme,amyloglucosidase (AMG). This is a feasible approach to increase dailyenergy gains and improve the health of malnourished children. Such anapproach would also be useful for treatment of congenital sucraseisomaltase syndrome (Gericke et al., 2016), functional bowel disorders(Henstrom et al., 2018; El-Chammas et al., 2017), small bowel bacterialovergrowth, radiochemotherapy induced mucositis and short-gut syndrome.For these purposes, the associated technical considerations towardoptimizing starch digestion using amyloglucosidase are addressed.Embodiments of the disclosure include daily AMG supplementation, forexample, with the overarching goal of improving growth and generalhealth of the afflicted.

In some embodiments, at least two ethanol washes are required tosignificantly decrease the crude AMG of toxin in which the processexploits the feature that the AMG is completely insoluble in ethanol anddeoxynivalenol is completely soluble. The knowledge of this dichotomy isnot obvious, since deoxynivalenol is seldom, if ever, tested for inproducts derived from A. niger; a totally unexpected finding and uniqueand useful to these purposes.

II. Amyloglucosidase (AMG) Compositions

The present disclosure includes AMG compositions that are specificallyformulated (and may also be referred to as AMG formulations). The AMGcompositions may be produced by methods described herein. In particularembodiments, the AMG formulations may or may not have a specific amountof AMG within. In certain embodiments, the AMG formulation is toxin-freeor is substantially reduced in the level of one or more toxins comparedto a standard or crude preparation. The toxin may be of any kind, but inspecific embodiments the toxin is deoxynivalenol (vomit toxin). Inparticular embodiments, the AMG formulation has both of (1) a particularamount of AMG; and (2) is toxin-free or substantially reduced in itslevel of toxin(s).

In particular cases, the level of AMG in the AMG formulation is equal toor greater than 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000;9,000; 10,000; 11,000; 12,000; 13,000; 14,000; 15,000; 16,000; 17,000;18,000; 19,000; 20,000; or greater unit releases of one gram of glucoseper hour (AGU). In specific cases, AMG is released at 5 The AMG may bereleased at 5,000-20,000; 5,000-15,000; 5,000-12,000; 5,000-10,000;5,000-9,000; 5,000-8,000; 5,000-7,000; 5,000-6,000; 6,000-20,000;6,000-15,000; 6,000-12,000; 6,000-10,000; 6,000-8,000; 7,000-20,000;7,000-15,000; 7,000-12,000; 7,000-10,000; 8,000-20,000; 8,000-15,000;8,000-12,000; 8,000-10,000; 6,000-10,000; 6,000-9,000; 6,000-8,000;6,000-7,000; 7,000-10,000; 7,000-9,000; 7,000-8,000; 8,000-10,000;8,000-9,000; 9,000-10,000; 10,000-20,000; 10,000-19,000; 10,000-18,000;10,000-17,000; 10,000-16,000; 10,000-15,000; 10,000-14,000;10,000-13,000; 10,000-12,000; 10,000-11,000; 11,000-20,000;11,000-19,000; 11,000-18,000; 11,000-17,000; 11,000-16,000;11,000-15,000; 11,000-14,000; 11,000-13,000; 11,000-12,000;12,000-20,000; 12,000-19,000; 12,000-18,000; 12,000-17,000;12,000-16,000; 12,000-15,000; 12,000-14,000; 12,000-13,000;13,000-20,000; 13,000-19,000; 13,000-18,000; 13,000-17,000;13,000-16,000; 13,000-15,000; 13,000-14,000; 14,000-20,000;14,000-19,000; 14,000-18,000; 14,000-17,000; 14,000-16,000;14,000-15,000; 15,000-20,000; 15,000-19,000; 15,000-18,000;15,000-17,000; 15,000-16,000; 16,000-20,000; 16,000-19,000;16,000-18,000; 16,000-17,000; 17,000-20,000; 17,000-19,000;17,000-18,000; 18,000-20,000; 18,000-19,000; or 19,000-20,000 AGU. Insome cases, the AMG formulation comprises approximately 20000 AGUactivity units per gram (2 capsules) or less dependent upon desireddilution.

In particular embodiments, the level of AMG in a formulation orcombination of formulations is greater than 20,000 AGU. The skilledartisan recognizes that lower levels of AMG in an AMG formulation may bemore suitable for use as a supplement, whereas higher levels of AMG inan AMG formulation may be more suitable for use in another purpose, suchas a medical food.

In certain embodiments, the AMG composition is formulated in a specificform, such as a tablet, pill, film, lozenge, powder, capsule, orcombination thereof. In particular cases the AMG formulation is acapsule. In cases wherein the AMG formulation is in a capsule, thecapsule may comprise at least about 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, or 10,000 or more AGU, particularly about 8000 AGU. Inother cases, an AMG formulation in a capsule comprises no more thanabout 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 ormore AGU. The skilled artisan recognizes that with capsule preparation(as an example) the enzyme content may be determined by the number oftimes that one admixes (for example, by spray) a purified, toxin-freeliquid containing the AMG to the excipient and allowing it to dry. Insome cases, after the drying step, in subsequent steps one could againadmix the toxin-free liquid containing the AMG with unpacked powder (theexcipient) to increase the concentration.

Nutraceutical compositions of the present disclosure (that may also bereferred to as medicinal food, medical food as defined in section 5(b)of the Orphan Drug Act (21 U.S.C. 360ee (b) (3)), food ingredient,dietary supplement, and/or drug preparation) comprise an effectiveamount of one or more AMG compositions dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a nutraceutical composition thatcontains at least one AMG composition or additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington: The Science and Practice ofPharmacy, 21^(st) Ed. Lippincott Williams and Wilkins, 2005,incorporated herein by reference.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The AMG composition may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it need to be sterile for such routes of administration asinjection. The present invention can be administered enterically,intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in creams, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The AMG composition may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present disclosure, the AMG compositionof the present disclosure suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present disclosure, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present disclosure, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the AMG compositions may be dispersed in asolution containing a lipid, dissolved with a lipid, emulsified with alipid, mixed with a lipid, combined with a lipid, covalently bonded to alipid, contained as a suspension in a lipid, contained or complexed witha micelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present disclosureadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In particular embodiments of the present disclosure, the AMGcompositions are formulated to be administered via an alimentary route.Alimentary routes include all possible routes of administration in whichthe composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, aluminum hydroxide, magnesium hydroxide, sodium bicarbonate,calcium carbonate, calcium phosphate, calcium silicate, calciumchloride, or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present disclosure mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

In particular embodiments of the AMG compositions, no stabilizers arepresent.

In particular embodiments, the AMG composition is combined with one ormore other therapeutic agents. An individual may receive the AMGcomposition in the same composition as the one or more other therapeuticagents, or the individual may receive them separately. In some cases,the one or more other therapeutic agents comprise an agent thatfacilitates digestion of biological molecules other than starch, such assugar, lactose, and so forth. In specific cases the one or more othertherapeutic agents comprise invertase, sucrose, lactase, xyloseisomerase (glucose isomerase), beta-galactosidase, or a combinationthereof. In other specific cases the one or more therapeutic agents thatconstitute type-2-histamine receptor antagonists (e.g., ranitidine HCl)or benzimidizoles, also known as proton-pump inhibitors (e.g.,omeprazole), or a combination thereof.

III. Methods of Producing Amyloglucosidase (AMG) Compositions

Methods of producing one or more AMG compositions are encompassed in thedisclosure. In particular embodiments, the method is performed for thepurpose of removing or reducing the level of one or more toxins. Themethod of producing toxin-free AMG compositions or substantially reducedlevels of toxin in AMG compositions may be performed with the intent ofremoving toxin(s) from commercially sourced raw/crude product (as anexample). Crude AMG may be produced from A. niger using wet submersionor solid state techniques, for example.

Embodiments of the disclosure include methods for producing purified AMGcompositions, including toxin-free AMG compositions or compositions havereduced levels of AMG compared to starting AMG compositions. In somecases the method steps comprise extracting a crude AMG composition inthe presence of an immiscible solvent and separating a purified AMGcomposition from the immiscible solvent. A crude AMG composition (whichmay be a solid) may be defined as one that has not been subjected to anextraction/purification step and/or it is a composition from whichtoxins and other impurities have not been removed intentionally.Although the immiscible solvent may be of a variety of suitable kinds,in specific embodiments the immiscible solvent is absolute ethanol. Inparticular embodiments, the solvent from which the purified AMGcomposition is separated comprises one or more toxins, including thedeoxynivalenol “vomit toxin” from the crude enzyme, as an example. Theextracting step may comprise vortexing (mixing) and/or blending thecrude AMG composition and the immiscible solvent.

The method may include isolating steps of any kind, but in specificcases the isolating of the retained, purified AMG composition comprisesspinning down the amyloglucosidase composition in a centrifuge orpermitting gravity separation to occur over prolonged time.

In the production methods, one or more steps may be repeated, includingat least repeating the extracting and/or isolating steps one or moretimes, for example.

Once the AMG composition has been isolated, it may be further modifiedor prepared, such as dissolving the retained AMG composition in asolvent (such as water) to form a solution or suspension. In at leastsome cases the solution or suspension may be filtered, centrifuged,and/or separated by gravity over time.

In some embodiments, the resultant AMG composition produced by methodsof the disclosure is formulated into a particular composition that is acapsule, tablet, pill, film, lozenge, powder, or combination thereof, asexamples. Aqueous AMG solution may be admixed with inert excipient, suchas calcium carbonate, calcium phosphate and calcium silicate or acombination thereof and dried to powder for mechanical or hand gelatinencapsulation.

In specific embodiments, AMG compositions of the disclosure are utilizedimmediately or they may be stored, such as under dry conditions, lowhumidity and room temperatures while avoiding excessive heat or cold. Itmay be transported prior to use.

IV. Alternative Method of Producing Amyloglucosidase (AMG) Compositions

In specific embodiments, crude AMG is supplied by a producing vendor inthe form of a particulate-free dark, semi-opaque liquid product ofAspergillus niger liquid-state fermentation that is intended forproduction of purified dry composition. The crude liquid AMG may containDON toxin and/or other non-detectable organic impurities includingsesquiterpenes and sesquiterpenoids. The sesquiterpenes andsesquiterpenoids are by nature, lipophillic and soluble in absoluteethanol. As supplied by the vendor, the crude liquid AMG may have anenzyme activity that approximates 1000 AGU per milliliter (1000 AGU permilliliter could be 100-500 AGU, 500-1000 AGU, 1000 to 1500 AGU,1500-2000 AGU, 2000-2500 AGU or 2500-3000 AGU) and must be in a dry formto facilitate purification of the final composition.

In specific embodiments, the purification process uses GoodManufacturing Practices and will take place in a controlled, lowhumidity clean room supplied by high efficiency particulate air filteredair (limit 0.3 micrometers) and includes several concentration stepsthat includes admixing crude liquid AMG to inert solid excipient, suchas a mixture calcium carbonate, calcium phosphate and calcium silicate(“excipient mixture”) to create a batter using a standard low-speedbakers mixer and stainless steel bowl or similar instrument. The batteris spread thinly over food-grade plastic wrap used as liner for largecookie sheets (i.e., sized 24×36 inches) to maximize evaporative surfaceareas and the batter is permitted in evaporate in extremely lowhumidity, clean room conditions at approximately 10-15 degrees untilvisibly dry. Alternatively, the crude liquid AMG may be applied to theexcipient mixture by aerosolizing the crude liquid AMG and spray-appliedit evenly to the excipient mixture layers contained by the cookiesheets. The admixture is composed of a ratio of 4 parts crude liquid AMGto 1 part excipient mixture and permitted to dry by evaporation over aperiod of 24-hours to 36-hours depending upon conditions to yield acrude composition. Dryness is determined by brittle nature of thecomposition and the lightening of the color from dark brown to light tanwith a dusty white. The dry crude composition is then recovered to aclean blender (such as Waring 023909) and pulverized to a fine powder,taking care not to heat the crude composition with prolonged high-speedpulverization and using short bursts of pulverizing activity is bestpractice. With the intention to concentrate the final composition toyield high enzymatic activity, additional crude liquid AMG may beadmixed again with the primary dry crude composition a ratio of 4 partscrude liquid AMG to 1 part excipient mixture and permitted to dry byevaporation over a period of 24-hours to 36-hours depending uponconditions to yield a secondary concentrated crude composition. Thisadmixing sub-process may be repeated to increase enzyme activity to thedegree that it becomes impractical to advance with purificationprocesses described below.

After repeat pulverization, the secondary concentrated crude compositionis aliquoted is small batches to high density, food-grade polypropylenecontainers (for example 1 liter capacity (for example Nalgene 2178-2025Tritan 32 oz W/M, Gray w/Blue Cap), for extraction (washing) to whichabsolute ethanol is added in a ratio of 1 part absolute ethanol (USPgrade) to 1 part secondary composition such that the combined volume ofthe container approximates 66 percent of capacity. The capacity of thecontainer should not exceed 75% total volume so as to permit excellentagitation during the extraction phase. In clean room conditions atapproximately 10-15 degrees the extraction phase is performed wherebythe secondary composition/ethanol mixture is gas-tight sealed and shakenvigorously at least 30 minutes using a commercial paint canshaker-mixer, such as Red Devil Paint Shaker: Multi-Size Case Shakermachine, 5 gal Container Size 5995PB, or similar device, that can acceptmultiple high density, food-grade polypropylene containers within anouter wrapping container used for consolidation and fire safety. Aftervigorous shaking (extraction/washing) the high density, food-gradepolypropylene containers are removed from the shaker machine and thecontents within the high density, food-grade polypropylene containersare permitted to separate by gravity (about 2 hours). The alcoholicsupernatant is then carefully aspirated to waste taking care to retainthe composition within the high density, food-grade polypropylenecontainers. To each high density, food-grade polypropylene containerscontaining the composition is added an equal volume of absolute ethanol(USP grade), whereupon the container are resealed and again, the highdensity, food-grade polypropylene containers are shaken vigorously atleast 30 minutes using a commercial paint can shaker-mixer, such as RedDevil Paint Shaker: Multi-Size Case Shaker machine, 5 gal Container Size5995PB, or similar device, that can accept multiple high density,food-grade polypropylene containers within an outer wrapping containerused for consolidation and fire safety. After vigorous shaking(extraction/washing) the high density, food-grade polypropylenecontainers are removed from the shaker machine and outer container andthe contents are permitted to separate by gravity (about 2 hours).Again, the alcoholic supernatant is then carefully aspirated to wastetaking care to retain the composition within the high density,food-grade polypropylene containers. The remaining composition isremoved from each high density, food-grade polypropylene container, thecomposition is thinly distributed on lined cookie sheets as aliquots andany residual ethanol is permitted to evaporate to dry in a safe,spark-free, clean low humidity room. When the composition aliquots aredry, the aliquots are pulverized, well mixed, and combined to bulkpackaging that includes heavy duty, food grade plastic bags (such asHefty Slider 2.5 Gallon Jumbo Storage Bags, Reynolds Consumer ProductUPC 700064844090), labeled and made ready for release testing anddistribution according to specifications. The composition may be dilutedwith excipients to yield a desired, lower enzymatic activity. AMGcompositions of the disclosure are utilized immediately or they may bestored, such as under dry conditions, low humidity and room temperatureswhile avoiding excessive heat or cold. It may be transported prior touse.

V. Methods of Optimizing Starch or Fructans Digestion

In particular embodiments, an effective amount of an AMG compositionencompassed by the disclosure is provided to an individual in needthereof, including an individual in need of optimizing or improvingstarch or fructans digestion. The individual may be in need of AMGsupplementation for any reason. In other cases, the individual may notbe in need of AMG supplementation but instead is taking AMGcomposition(s) of the disclosure as a routine practice, as maintenance,or as prevention of a need for AMG supplementation. In specificembodiments, an individual has a medical condition associated withinsufficient disaccharidase levels or insufficient starch digestion orinsufficient fructans digestion, for example. In specific embodiments,the individual has congenital sucrase isomaltase deficiency syndrome,celiac disease, parasitic infestation disease, viral gastroenteritis,mucosal injury from infectious gastroenteritis, small bowel mucositis, afunctional bowel disorder, functional bowel syndrome, functionalgastroduodenal disorders, and/or protein calorie (energy) malnutrition.Although the individual may be malnourished, in some cases theindividual is not malnourished.

In cases wherein the AMG composition is utilized for functional boweldisorders, the individual may have difficulty with one or more of thefollowing: (1) the movement of food and waste through the GI tract; (2)heightened experience of pain in the internal organs; (3) changes in thegut's immune defenses; (4) changes in the community of bacteria in thegut; and (5) changes in how the brain sends and receives signals fromthe gut.

In cases wherein the AMG composition is employed for functionalgastroduodenal disorders, the individual may be experiencing one or moreof the following: (1) functional dyspepsia (FD) including postprandialdistress syndrome (PDS) and epigastric pain syndrome (EPS); (2) belchingdisorders including excessive gastric and supragastric belching; (3)chronic nausea and vomiting disorders including chronic nausea vomitingsyndrome (CNVS), cyclic vomiting syndrome (CVS) and “cannabinoidhyperemesis syndrome” (CHS); and (4) rumination syndrome.

In specific cases, AMG is utilized for hydrolysis of fructans (polymersof fructose found in many fruits and vegetables). An individual in needof improved digestion of fructans may be provided an effective amount ofAMG. In a specific case, an individual is sensitive to FODMAPs(“fermentable oligo-, di-, mono-saccharides and polyols”) and isprovided an effective amount of AMG. The individual may have fructanintolerance. The individual may be administered AMG at the same time as,before, and/or after the consumption of FODMAPs. In some cases, inaddition to taking an effective amount of AMG they may be controllingthe consumption of foods or beverages high in fructose. The individualmay have fructan maldigestion, such as with consuming prunes and/orraisins.

An individual that consumes or otherwise intake or be administered maybe of any gender, race, or age. The individual may be an infant, child,adolescent, or adult, including a gestating adult. The individual may beadministered the AMG composition(s) once or more than once. Followingadministration, such as in a routine manner, improvement of at least onesymptom of a medical condition may improve and the individual may or maynot continue to receive the AMG composition(s) following this. Theindividual may be administered the AMG composition daily, 2-3-4-5-6times daily, weekly, monthly, yearly, etc. The individual may or may nottake the AMG composition(s) with food. In some cases, the individual maytake the AMG composition before consumption of food and/or beverage,during consumption of food (including starch) and/or beverage, and/orfollowing consumption of food and/or beverage. In cases wherein the AMGcomposition is taken before the consumption of food, the composition maybe microencapsulated for delayed release (or the composition may bemicroencapsulated for any other reason). In particular cases, the foodcomprises one or more starches, such as cassava, millet, sorghum, rice,wheat, potato, tapioca, corn, rye, oats, a combination thereof, and soforth. In particular embodiments, the AMG composition is containedwithin a food or beverage.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Amyloglucosidase as a Nutraceutic for Maldigestion andMalnutrition

Nutritional substances were considered that would be useful in severalsymptomatic populations, including functional bowel disorder, functionalbowel syndrome, functional gastroduodenal disorders, congenitalsucrase-isomaltase insufficiency, iatrogenic mucositis, undernourishedlactating women, and undernourished children, for example. In particularembodiments, a dietary product is produced that alleviates one or moresymptoms of disaccharidase insufficiency from any cause, for example.

Considering that SGLT-1 transporters are relatively conserved, even inthe face of severe malnutrition, supplementing or replacing endogenousisomaltase with amyloglucosidase is feasible, as in the case offunctional bowel disorder, functional bowel syndrome, functionalgastroduodenal disorders, Congenital Sucrase-Isomaltase Deficiency(CSID), mucositis, and small-bowel bacterial overgrowth that is expectedto diminish typical malabsorption symptoms caused by excessive colonicfermentation.

Infectious diarrhea that causes significant intestinal mucosal injuryand loss of naturally inherent mucosa-bound disaccharidases(sucrase-isomaltase and maltase glucoamylase) often leads to variousdegrees of malnutrition. Feeding malnourished pediatric patients offersa great challenge. Such a treatment is useful for malnourished children(for example, in sub-Saharan nations) to allow them to efficientlydigest their carbohydrate sparse foodstuffs (e.g., sorghum and millet),including while recovering from various illnesses that have compromisedtheir normal digestive capacity. Marasmoid patients have difficultydigesting carbohydrates, which is typically the most abundant (common)food stuff. If digestion of ingested grains could be accelerated, thenutritional benefits could be realized.

In one embodiments, amyloglucosidase is selected for subsequent testingin humans, with or without invertase and/or xylose isomerase, as amedicinal food product (a nutraceutical or food supplement). To meetthis objective, several commercial grade samples of amyloglucosidase andinvertase and/or xylose isomerase were selected for purity and activityusing the following procedures: enzyme concentration by Lowry method,enzyme characterization by SDS-PAGE methodology, enzyme purity by thinlayer chromatography screen for low molecular weight contaminants, andenzyme activity by rate of glucose production. The results of thesestudies facilitated selection of a primary and secondaryamyloglucosidase candidate from which a refined material could beencapsulated and repackaged to be used as a nutraceutical or medicinalfood.

In another embodiment there is identification of an establishedmanufacturer to prepare and encapsulate amyloglucosidase for humanconsumption. In a specific embodiment, one tests amyloglucosidasesupplement for activity in people with congenital Sucrase-Isomaltasedeficiency syndrome and demonstrate symptomatic relief. In anotherembodiment, amyloglucosidase supplement is tested for activity in peoplewith functional bowel disorder and demonstrates symptomatic relief. Onecan also test amyloglucosidase supplementation activity in children withprotein-calorie malnutrition and demonstrate increased caloric yield bydemonstrating weight gain and improved health and well-being orresolution of diarrhea, for example.

Methods Summary

The following is a description of an embodiment of methods of thedisclosure.

Sample Preparations: Following receipt of the test article, the severalcandidate enzyme samples were annotated and given a tracing number.Subsequently, a 50 mL solution was prepared from powder candidate enzymeusing standard sodium citrate buffer [10 mM, pH 4.5, Fisher S279; SigmaC0759, de-ionized water (Barnstead NanoPure II)] at a candidate enzymeconcentration of 5 mg/mL (Robyt and White, 1987; Standard Solutions).Subsequently, half (25 mL) was dialyzed using 50 kD MWCO cellulose estermembrane tubing (part #131384. Lot #3272198; Spectrum Labs, RanchoDominquez, C A) in citrate buffer (dialysate) to remove all free glucoseand low molecular weight substance that might interfere with subsequentassays. The test article samples underwent a Lowry assay to determineprotein concentration and diluted proportionately with standard sodiumcitrate buffer to yield a concentration of 1 mg/mL for further testing,unless otherwise indicated. In a similar fashion, a candidate crudeenzyme provided in liquid form by the manufacturer, was directlydialyzed before determination of protein concentration and, dependingupon the determined protein concentration, diluted proportionately withstandard sodium citrate buffer to yield a concentration of 1 mg/mL.

Protein Concentration: The Lowry assay is the method of choice foraccurate protein determination for cell fractions, chromatographyfractions, and enzyme preparations (Robyt and White, 1987; Quantitativedetermination of proteins). Under alkaline conditions, the divalentcopper ions forms a complex with peptide bonds in which it is reduced toa monovalent ion. Monovalent copper ion and the radical groups oftyrosine, tryptophan, and cysteine reactwith Folin reagent to produce anunstable product that becomes reduced to molybdenum/tungsten blue.Sample intensity is measured using an IR-spectrophotometer and resultsare compared against a standard reference curve to determine sampleprotein concentration.

SDS-PAGE: Samples underwent sodium-dodecyl-sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) analysis to affirm that the product receivedwas as stated and relatively void of extraneous proteins (Robyt andWhite, 1987; Gel electrophoresis).

Thin Layer Chromatography (TLC): Mycotoxins may be surveyed using gaschromatography (GC), high performance liquid chromatography (HPLC) andthin-layer chromatographic (TLC) methodologies. TLC is a technique usedfor the identification or separation of compounds in mixtures (Robyt andWhite, 1987; Thin-layer chromatography). It is routinely used to monitorthe consumption of starting material in bioreactors and to observe forthe appearance of desired products and problematic contaminants, such aspesticides and toxins (Ambrus et al, 2005). Unwanted organic productsproduced by Aspergillus species may rarely include toxic contaminantssuch as mycotoxins (ochratoxins) and aflatoxins. Mycotoxins are lowmolecular weight secondary fugal metabolites that may result in acute orchronic disease. Of particular concern is deoxynivalenol (“DON-V”or“vomit toxin”; C15H20O6/296.32 g·mol-l), a sesquiterpenoidtrichothecene and it is among the most often encountered mycotoxins (Linet al, 1998). It is typically found in grains contaminated by Fusariumspecies. Hundreds of structurally related, LMW mycotoxins are known toexist and epidemiological studies have shown an association ofgastrointestinal distress. DON is not known to be carcinogenic, butacute toxicity is potent and the FDA has placed a limit of 1 ppmrestriction on it. The most convenient method to test for deoxynivalenolis by use of fluorescent immunochemistry, for which commercial test kitsare readily available.

Estimation of Enzyme Activity by Rate of Glucose Production: Samplesunderwent activity testing to assess which candidate sample enzyme wasthe most potent for hydrolyzing starch and dextrins and to determine ifany of the AMG samples had any a-D-glucopyranosyl-(12)-β-D-fructofuranoside hydrolytic activity (Robyt and White, 1987;Determination of enzyme activity). A panel of standard substrates wastested, and results were reported in terms of the rate of glucoseproduction as measured by a glucometer (e.g., Analox GM9, (Robyt andWhite, 1987; Quantitative determination of carbohydrates). Analoxanalyzers are based on the principle of enzymatic oxygen uptake of theanalyte (sample to be measured) using specific oxidoreductase anddehydrogenase enzymes (reagent consumable) and are used in biotechnologyapplications (Cass et al, 1984). The instrument uses a highly accurateelectrochemical probe to measure oxygen uptake. Example equation(sample+reagent consumable): [β-D-GLUCOSE+O2-glucose oxidase→D-GLUCONICACID-H₂O₂] The change in oxygen ion concentration is measured by theinstrument and change in electrical potential correlates with glucoseconcentration. Traditional, alternative colorimetric methods are alsocontemplated.

Examples Of Results

Protein Identification by SDS-PAGE Methodology

The molecular weight and purity of candidate amyloglucosidase can beestimated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) techniques. The mobility of an enzyme in a gel is influencedby the size and charge of the molecule; therefore samples are usuallytreated to have a uniform charge, so electrophoretic mobility inSDS-PAGE depends mainly on molecular weight size. PAGE is carried out inthe presence of an anionic detergent, SDS, and a reducing agentmercaptoethanol (BME). SDS disrupts the secondary, tertiary andquaternary structure of the protein to produce a linear polypeptidechain coated with negatively charged SDS molecules and BME contributeswith protein denaturation by reducing all disulfide bonds. Conversely,native PAGE-enzymes may be prepared in a non-reducing/non-denaturingsample buffer (SDS-free or BME-free) and in the gel, which shouldmaintain both the enzymes' secondary structure and native charge densityto determine the aggregation state. This permits visualization ofmultiple bands if the target enzyme sample has polymerized forms.SDS-PAGE will not demonstrate the presences of LMW non-protein toxins.The SDS-PAGE results for AMG samples 26252, 26454, 26456, 26457, 2645926461 26424A, and 26424B, along with Sigma references for A. niger andA. rhizopus, are shown (FIG. 2A).

Estimation of Purity by Thin-Layer Chromatography

Thin layer chromatography (TLC) is a useful technique for the separationand identification of compounds in mixtures. TLC is used routinely tofollow the progress of reactions by monitoring the consumption ofstarting materials and observing for the appearance of products.Commercial applications of TLC include the analysis of aberrantsubstances to establish purity or identity of the components, andanalysis of foods to determine the presence of other compounds such asflavonoids, pesticides and possibly contaminants includingsesquiterpenoid trichothecenes (Eppley at al., 1984). See FIGS. 3-4 .

Estimation of Enzyme Activity by Rate of Glucose Production

One approach to the determination of enzyme activity is to estimate therate of glucose production from observations made on a controlledsubstrate reaction between enzymes and oligosaccharides (dextrins) orstarches. The assessment of glucose production involves the use of testor HPLC-grade reference enzymes to hydrolyze oligosaccharides orstarches, and indirect measurements of resultant free glucose can bedone. The traditional technical approach relies upon a glucose oxidationreaction that ultimately yields a reactive species that effects acolorimetric change that, in turn, correlates with glucose concentrationwhen compared with standards and control reagents. A contemporaryapproach relies upon a change in oxygen ion concentration thatcorrelates with glucose concentration. In the later scenario, oxygenconsumption during glucose oxidation is detected by use of a Clark-typeamperometric, platinum oxygen electrode. This is the same highlyaccurate method upon which blood gas analyzers are based. Other glucoseassays rely upon sample hydrolysis of p-nitrophenyl-α-glucopyraniside(PNGP; C12H15NO8; Molecular Weight 301.25) to p-nitrophenol (PNGP) andglucose and colorimetric measurement of PNGP at pH 4.3 and 50° C. Theelectrochemical (oxygen consumption) measurement of glucose productionclosely equates to p-nitrophenyl since the molecule is hydrolyzed in a1:1 ratio. The electrochemical technology used in these experiments isexceptionally fast, with results available in less than 20 seconds aftersample injection. This timely approach prevents overestimation of enzymeactivity and is remarkably free from the interference problemsassociated with antiquated PNGP optical techniques. As such, testenzymes, for which activity is undetermined, can be assayed using thesesemi-micro quantitative analyses. The work described herein relates tothe quality (activity) of the particular food-grade, fungal-derivedenzymes, such as amyloglucosidase produced by molecular biology ormicrobial culture, to release glucose from target starch oroligosaccharide substrates in the human alimentary tract.

Amyloglucosidase has broader activity than mammalian physiologicalenzymes, and alpha amylase is not needed to liberate glucose fromstarch. Amyloglucosidase is a robust candidate therapeutic agent and isprobably active in the stomach, especially when food ingestion, takenconcomitantly, raises pH. Invertase, an enzyme used to hydrolyzedsucrose, may be assayed in a similar way. Several candidate digestiveenzyme samples (amyloglucosidase or invertase) were tested. The amountof enzyme was adjusted based upon determined protein concentrationsunless otherwise specified. Test conditions included use of 10 mMcitrate buffer at pH 4.5 and 37 C. Results are reported regardingmilligrams glucose produced periodically (over 60 minutes) and convertedto USP activity units consistent with USP Dietary SupplementsCompendium, (the United States Pharmacopoeia Convention, Inc.,Rockville, Md., USP/DSC 2nd ed., 2012) Appendix V, Enzyme Assays pg.1727-1759. One unit of glucoamylase activity is defined as the amount ofglucoamylase that will hydrolyze 0.1 μmol/min of maltose under theconditions of the assay (same as produce 0.2 μmol/min of glucose). Note:in summary, the calculations rely on the peak delta milligrams glucoseproduced per minute, which is usually the first 20 minutes of the assay.The minute-mean is determined and milligrams are converted tomicromoles. The AMG activity is expressed as International Units (IU)per milligram enzyme. HPLC-grade analytical enzymes were used forcomparative purposes.

TABLE 1 Enzyme Activity: Data Summary Accession # Enzyme Name SupplierActivity (Qual) Comments 26424B & 26437 Amyloglucosidase UltrabiologicsExcellent~0.5 Powder AGU/15 mg protein 26452 Glucoamylase NECExcellent~0.5 Powder AGU/15 mg protein 26454 Amyloglucosidase CreativeBiomart Not Performed Protein Minimal 26456 Amyloglucosidase Bio-CatPoor Powder 26457 Glucoamylase NEC Fair Powder Excellent 26461Amyloglucosidase Creative Enzymes Fair Black liquid

TABLE 2 Enzyme Activity (see FIGS. 5-8 for the following tables)Reaction Vial # 0 10 20 30 40 50 60 end Sample: NEC GLUCOAMYLASE 26452 11 mg/mL 26452 7.6 ND ND ND ND ND ND 11.6 Glucoamylase control, nosubstrate 2 1 mg/mL 26452 19.5 526.0 1052.0 1023.6 1003.6 967.8 948.8 NDGlucoamylase and 1% Maltose 3 1 mg/mL 26452 14.8 19.7 25.2 31.2 38.545.0 56.6 ND Glucoamylase and 1% Sucrose 4 1 mg/mL 26452 110.3 526.01052.0 1052.0 1052.0 1052.0 1052.0 ND Glucoamylase and 1% Rice Starch 51 mg/mL 26452 66.4 526.0 1031.0 962.4 942.4 913.8 900.2 ND Glucoamylaseand 1% Cornstarch 6 1% Maltose control, 4.0 ND ND ND ND ND ND 10.3 noenzyme 7 1% Sucrose control, 5.0 ND ND ND ND ND ND 7.8 no enzyme 8 1%Rice Starch 8.1 ND ND ND ND ND ND 13.2 control, no enzyme 9 1%Cornstarch 0.9 ND ND ND ND ND ND 1.9 control; no enzyme 10 25 mg/dLGlucose 23.1 ND ND ND ND ND ND 22.7 standard 11 100 mg/dL 97.0 ND ND NDND ND ND 94.3 Glucose Standard 12 250 mg/dL 249.2 ND ND ND ND ND ND247.9 Glucose Standard 13 400 mg/dL 397.7 ND ND ND ND ND ND 394.0Glucose Standard Sample: Bio-Cat Amyloglucosidase 26456 1 1 mg/mL AMG,10.2 ND ND ND ND ND ND 13.3 no substrate 2 1 mg/mL AMG, 14.6 15.9 23.131.5 41.3 49.4 56.6 ND 1 mg/mL sucrose 3 1 mg/mL AMG, 17.8 235.7 220.6212.1 212.2 204.9 209.8 ND 1 mg/mL maltose 4 1 mg/mL AMG and 21.1 244.9228.2 213.4 210.5 205.0 202.2 ND 1 mg/mL corn starch 5 1 mg/mL AMG and104.9 340.5 316.9 300.2 293.1 290.4 288.6 ND 1 mg/mL rice Starch 6 1mg/mL AMG and 138.5 409.0 409.0 475.6 472.2 458.8 454.8 ND 1 mg/mLpotato starch 7 1 mg/mL Sucrose 10.3 ND ND ND ND ND ND 1.1 control, noenzyme 8 1 mg/mL Maltose, 2.1 ND ND ND ND ND ND 2.5 no enzyme 9 1 mg/mLcorn Starch, 1.1 ND ND ND ND ND ND 2.1 no enzyme 10 1 mg/mL rice starch,4.4 ND ND ND ND ND ND 5.8 no enzyme 11 1 mg/mL potato starch 1.6 ND NDND ND ND ND 2.1 control, no enzyme 12 25 mg/dL glucose 21.9 ND ND ND NDND ND 22.4 standard 13 100 mg/dL glucose 96.2 ND ND ND ND ND ND 96.3standard 14 400 mg/dL Glucose 379.9 ND ND ND ND ND ND 387.5 StandardSample: NEC Amyloglucosidase 26457 1 1 mg/mL 26457 24.3 ND ND ND ND NDND 20.7 AMG control, no substrate 2 1 mg/mL 26457 19.0 21.3 24.0 27.531.6 36.0 40.4 ND AMG and 1 mg/mL Sucrose 3 1 mg/mL 26457 27.9 239.1228.3 217.3 216.8 214.5 213.5 ND AMG and 1 mg/mL Maltose 4 1 mg/mL 26457AMG 26.3 252.0 233.8 220.1 210.9 208.7 207.6 ND and 1 mg/mL Corn starch5 1 mg/mL 26457 AMG 110.0 343.0 320.8 304.3 295.5 290.8 292.0 ND and 1mg/mL Rice Starch 6 1 mg/mL 26457 AMG 158.3 407.6 407.6 455.4 446.4438.8 435.4 ND and 1 mg/mL Potato Starch 7 1 mg/mL Sucrose 0.3 ND ND NDND ND ND 2.2 control, no enzyme 8 1 mg/mL Maltose 1.0 ND ND ND ND ND ND4.1 control, no enzyme 9 1 mg/mL Corn Starch 1.3 ND ND ND ND ND ND 3.7control, no enzyme 10 1 mg/mL Rice starch 4.6 ND ND ND ND ND ND 6.4control, no enzyme 11 1 mg/mL Potato starch 2.7 ND ND ND ND ND ND 3.2control, no enzyme 12 25 mg/dL Glucose 24.2 ND ND ND ND ND ND 22.6standard 13 100 mg/dL Glucose 95.2 ND ND. ND ND ND ND 95.6 Standard 14250 mg/dL Glucose 243.1 ND ND ND ND ND ND 249.3 Standard 15 400 mg/dLGlucose 395.5 ND ND ND ND ND ND 397.5 Standard Sample: Creative EnzymesAmyloglucosidase 26461 1 1 mg/mL 26461 0.1 ND ND ND ND ND ND 0.1 AMGcontrol, no substrate 2 1 mg/mL 26461 49.4 140.7 132.6 127.1 123.5 122.9121.5 ND AMG and 1 mg/mL Sidma Potato starch 3 1 mg/mL 26461 15.7 157.3164.5 161.3 159.6 158.7 160.6 ND AMG and 1 mg/mL Sigma Corn starch 4 1mg/mL 26461 24.3 71.2 65.4 64.0 63.1 63.8 63.3 ND AMG and 1 mg/mLDextrin from Corn 5 1 mg/mL Sigma Potato 6.5 ND ND ND ND ND ND ND Starchcontrol, no enzyme 6 1 mg/mL Sigma 1.9 ND ND ND ND ND ND 4.6 Corn Starchcontrol, no enzyme 7 1 mg/mL Dextrin 1.6 ND ND ND ND ND ND 3.5 from corncontrol; no enzyme 8 25 mg/dL Glucose 22.0 ND ND ND ND ND ND 22.8standard 9 100 mg/dL Glucose 94.4 ND ND ND ND ND ND 94.6 Standard 10 250mg/dL Glucose 242.5 ND ND ND ND ND ND 241.9 Standard 11 400 mg/dLGlucose 390.2 ND ND ND ND ND ND 392.1 Standard

Example 2 Processed Ultrabiologic Amyloglucosidase (AMG)/Sorghum StarchActivity Experiment. 31 Jul. 2017 Aro/Ma

Goal: To compare between AMG activity after purifying and concentrating(decrease volume 50%) suspension prepared under new ethanol extractionprotocol, described elsewhere herein, and 500 mg AMG capsules activity,1 ml of the AMG suspension mentioned above to 500 mg of excipient B,(Calcium phosphate 10 g, Calcium silicate 10 g, Calcium Carbonate 80 g).

Given: AMW of glucose is 180.156 Daltons and 1 mg=0.0055 mmol

1 AGU (of AMG) unit activity=0.2 mol glucose released/minute (0.1 μmolmaltose equivalent (starch) hydrolyzed)

Methods Summary: Mix 75 grams crude Ultrabiologic amyloglucosidasepowder (Lot #5220511016, Exp 2.2016, BCM #700126437b) in milliQ water(qs to 250 mL). Mix well using blender (45′), then spin out crudematter, coarse and fine vacuum filter (Whatman #1, and Millipore 0.45 μMwith Whatman 42 pre-filter), and spin concentrate to 50% volume usingMillipore Centriprep 30K MWCO. Aliquot for assay of resultant lightbrown suspension. Test for absence of deoxynivalenol (Don-V) toxin.

Timed Assay (FIG. 9 ):

A) 1.00 mL AMG: 99.00 mL Sorghum (20 mg/mL) to deliver 5.0 μL to AnaloxGM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

B) 1 AMG capsule: 99.00 mL Sorghum (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

Data: Don-V toxin NEGATIVE

A) For 1 ml AMG suspension:

Δ Glucose increase at 1 minute=+102.9 mg/dL

Δ Glucose increase at 2.5 minutes=+122.0 mg/dL

Δ Glucose increase at 5 minutes=+131.7 mg/dL

Δ Glucose increase at 10 minutes=+121.7 mg/dL

B) For 1 AMG Capsule:

Δ Glucose increase at 1 minute=+40.9 mg/dL

Δ Glucose increase at 2.5 minutes=+66.4 mg/dL

Δ Glucose increase at 5 minutes=+73.6 mg/dL

Δ Glucose increase at 10 minutes=error reported

Calculations: A) For AMG suspension:

Mean 1 minute Δ Glucose increase at 5 minutes+26.34 mg/dL equivalent for+263.4 mg/L

1 mL AMG liberated+263.4 mg/min for 1.463 mmol/min

1.463 mmol×1000=1463 μmol

Conversion of μmol to AGU: 1463 μmol glucose/0.2 (for maltoseequivalents)/0.1=7315 AGU B) For AMG Capsule:

MEAN 1 minute Δ glucose increase at 5 minutes+14.72 mg/dL equivalent for+147.2 mg/L

1 mL AMG liberated+147.2 mg/min for 0.8096 mmol/min

0.8096 mmol×1000=809.6 μmol

Conversion of μmol to AGU: 809.6 μmol glucose/0.2 (for maltoseequivalents)/0.1=4048 AGU

Discussion: 1 mL processed AMG suspension has enzyme activity of 7315AGU when hydrolyzing Sorghum starch. While AMG capsule (PRODUCT) hasenzyme activity of 4048 AGU when hydrolyzing Sorghum starch.

Conclusion: 1 round of concentration in specific embodiments issufficient since 1 mL of PRODUCT will be absorbed to 500 mg excipientand dried providing 4048 AGU per capsule. There was a 45% loss in AMGactivity on Sorghum starch upon encapsulating with type B calciumexcipient.

Example 3 Processed Ultrabiologic Amyloglucosidase (AMG)/Rice StarchActivity Experiment. 31 Jul. 2017 ARO/MA

Goal: To compare between AMG activity after purifying and concentrating(decrease volume 50%) suspension prepared under new protocol, describedelsewhere, and 500 mg AMG capsules activity, 1 ml of the AMG suspensionmentioned above to 500 mg of excipient B, (Calcium phosphate 10 g,Calcium silicate 10 g, Calcium Carbonate 80 g).

Given: AMW of glucose is 180.156 Daltons and 1 mg=0.0055 mmol

1 AGU (of AMG) unit activity=0.2 μmol glucose released/minute (0.1 μmolmaltose equivalent (starch) hydrolyzed)

Methods Summary: Mix 75 grams crude Ultrabiologic amyloglucosidasepowder (Lot #5220511016, Exp 2.2016, BCM #700126437b) in milliQ water(qs to 250 mL). Mix well using blender (45′), then spin out crudematter, coarse and fine vacuum filter (Whatman #1, and Millipore 0.45 μMwith Whatman 42 pre-filter), and spin concentrate to 50% volume usingMillipore Centriprep 30K MWCO. Aliquot for assay of resultant lightbrown suspension. Test for absence of deoxynivalenol (Don-V) toxin.

Timed Assay (FIG. 10 ):

A) 1.00 mL AMG: 99.00 mL Rice starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

B) 1 AMG capsule: 99.00 mL Rice starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

Data: Don-V toxin NEGATIVE

A) For 1 ml AMG suspension:

Δ Glucose increase at 1 minute=+84.9 mg/dL

Δ Glucose increase at 2.5 minutes=+89.7 mg/dL

Δ Glucose increase at 5 minutes=+123 mg/dL

Δ Glucose increase at 10 minutes=+143 mg/dL

B) For 1 AMG Capsule:

Δ Glucose increase at 1 minute=+48.5 mg/dL

Δ Glucose increase at 2.5 minutes=+69.9 mg/dL

Δ Glucose increase at 5 minutes=+78.7 mg/dL

Δ Glucose increase at 10 minutes=+81.6 mg/dL

Calculations: A) For AMG suspension:

Mean 1 minute Δ Glucose increase at 5 minutes+24.58 mg/dL equivalent for+245.8 mg/L

1 mL AMG liberated+245.8 mg/min for 1.3519 mmol/min

1.3519 mmol×1000=1351.9 μmol

Conversion of μmol to AGU: 1351.9 μmol glucose/0.2 (for maltoseequivalents)/0.1=6759 AGU B) For AMG Capsule:

MEAN 1 minute Δ glucose increase at 5 minutes+15.75 mg/dL equivalent for+157.5 mg/L

1 mL AMG liberated+157.5 mg/min for 0.8741 mmol/min

0.8741 mmol×1000=874.1 μmol

Conversion of μmol to AGU: 874.1 μmol glucose/0.2 (for maltoseequivalents)/0.1=4370 AGU

Discussion: 1 mL processed AMG suspension has enzyme activity of 6759AGU when hydrolyzing Sorghum starch. While AMG capsule (PRODUCT) hasenzyme activity of 4370 AGU when hydrolyzing Sorghum starch.

Conclusion: 1 round of concentration in specific embodiments issufficient since 1 mL of PRODUCT will be absorbed to 500 mg excipientand dried providing 4370 AGU per capsule. There was a 35% loss in AMGactivity on rice starch upon encapsulating with type B calciumexcipient.

Example 4 Processed Ultrabiologic Amyloglucosidase (AMG)/Wheat StarchActivity Experiment. 31 Jul. 2017 ARO/MA

Goal: To compare between AMG activity after purifying and concentrating(decrease volume 50%) suspension prepared under new protocol, describedelsewhere, and 500 mg AMG capsules activity, 1 ml of the AMG suspensionmentioned above to 500 mg of excipient B, (Calcium phosphate 10 g,Calcium silicate 10 g, Calcium Carbonate 80 g).

Given: AMW of glucose is 180.156 Daltons and 1 mg=0.0055 mmol

1 AGU (of AMG) unit activity=0.2 μmol glucose released/minute (0.1 μmolmaltose equivalent (starch) hydrolyzed)

Methods Summary: Mix 75 grams crude Ultrabiologic amyloglucosidasepowder (Lot #5220511016, Exp 2.2016, BCM #700126437b) in milliQ water(qs to 250 mL). Mix well using blender (45′), then spin out crudematter, coarse and fine vacuum filter (Whatman #1, and Millipore 0.45 μMwith Whatman 42 pre-filter), and spin concentrate to 50% volume usingMillipore Centriprep 30K MWCO. Aliquot for assay of resultant lightbrown suspension. Test for absence of deoxynivalenol (Don-V) toxin.

Timed Assay (FIG. 11 ):

A) 1.00 mL AMG: 99.00 mL Wheat starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

B) 1 AMG capsule: 99.00 mL Wheat starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

Data: Don-V toxin NEGATIVE

A) For 1 ml AMG suspension:

Δ Glucose increase at 1 minute=+91.50 mg/dL

Δ Glucose increase at 2.5 minutes=+130.6 mg/dL

Δ Glucose increase at 5 minutes=+177.2 mg/dL

Δ Glucose increase at 10 minutes=+226.0 mg/dL

B) For 1 AMG Capsule:

Δ Glucose increase at 1 minute=+28.2 mg/dL

Δ Glucose increase at 2.5 minutes=+41.1 mg/dL

Δ Glucose increase at 5 minutes=+48.1 mg/dL

Δ Glucose increase at 10 minutes=+65.3 mg/dL

Calculations: A) For AMG suspension:

Mean 1 minute Δ Glucose increase at 5 minutes+35.44 mg/dL equivalent for+354.4 mg/L

1 mL AMG liberated+354.4 mg/min for 1.9492 mmol/min

1.9492 mmol×1000=1949.2 μmol

Conversion of μmol to AGU: 1949.2 μmol glucose/0.2 (for maltoseequivalents)/0.1=9746 AGU B) For AMG Capsule:

MEAN 1 minute Δ glucose increase at 5 minutes+9.62 mg/dL equivalent for+96.2 mg/L

1 mL AMG liberated+96.2 mg/min for 0.5291 mmol/min

0.5291 mmol×1000=529.1 μmol

Conversion of μmol to AGU: 529.1 μmol glucose/0.2 (for maltoseequivalents)/0.1=2645 AGU

Discussion: 1 mL processed AMG suspension has enzyme activity of 9746AGU when hydrolyzing Sorghum starch. While AMG capsule (PRODUCT) hasenzyme activity of 2645 AGU when hydrolyzing Sorghum starch.

Conclusion: 1 round of concentration in specific embodiments issufficient since 1 mL of PRODUCT will be absorbed to 500 mg excipientand dried providing 2645 AGU per capsule. There was a 73% loss in AMGactivity upon encapsulating with type B calcium excipient.

Example 5 Processed Ultrabiologic Amyloglucosidase (AMG)/Maize StarchActivity Experiment

Goal: To compare between AMG activity after purifying and concentrating(decrease volume 50%) suspension prepared under new protocol, describedelsewhere, and 500 mg AMG capsules activity, 1 ml of the AMG suspensionmentioned above to 500 mg of excipient B, (Calcium phosphate 10 g,Calcium silicate 10 g, Calcium Carbonate 80 g).

Given: AMW of glucose is 180.156 Daltons and 1 mg=0.0055 mmol

1 AGU (of AMG) unit activity=0.2 μmol glucose released/minute (0.1 μmolmaltose equivalent (starch) hydrolyzed)

Methods Summary: Mix 75 grams crude Ultrabiologic amyloglucosidasepowder (Lot #5220511016, Exp 2.2016, BCM #700126437b) in milliQ water(qs to 250 mL). Mix well using blender (45′), then spin out crudematter, coarse and fine vacuum filter (Whatman #1, and Millipore 0.45 μMwith Whatman 42 pre-filter), and spin concentrate to 50% volume usingMillipore Centriprep 30K MWCO. Aliquot for assay of resultant lightbrown suspension. Test for absence of deoxynivalenol (Don-V) toxin.

Timed Assay (FIG. 12 ):

A) 1.00 mL AMG: 99.00 mL Maize (20 mg/mL) to deliver 5.0 μL to AnaloxGM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 5 and 10 minute elapsed reaction time.

B) 1 AMG capsule: 99.00 mL Maize (20 mg/mL) to deliver 5.0 μL to AnaloxGM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

Data: Don-V toxin NEGATIVE

A) For 1 ml AMG suspension:

Δ Glucose increase at 1 minute=+227.4 mg/dL

Δ Glucose increase at 5 minutes=+510.3 mg/dL

Δ Glucose increase at 10 minutes=+530.9 mg/dL

B) For 1 AMG Capsule:

Δ Glucose increase at 1 minute=+136.9 mg/dL

Δ Glucose increase at 5 minutes=+307.7 mg/dL

Δ Glucose increase at 10 minutes=+383.3 mg/dL

Calculations: A) For AMG suspension:

Mean 1 minute Δ Glucose increase at 5 minutes+102.06 mg/dL equivalentfor +1020.6 mg/L

1 mL AMG liberated+1020.6 mg/min for 5.664 mmol/min

5.664 mmol×1000=5664 μmol

Conversion of μmol to AGU: 5664 μmol glucose/0.2 (for maltoseequivalents)/0.1=28321 AGU B) For AMG Capsule:

MEAN 1 minute Δ glucose increase at 5 minutes+61.54 mg/dL-equivalent for+615.4 mg/L

1 mL AMG liberated+615.4 mg/min for 3.4154 mmol/min

3.4154 mmol×1000=3415.4 μmol

Conversion of μmol to AGU: 3415.4 μmol glucose/0.2 (for maltoseequivalents)/0.1=17077 AGU

Discussion: 1 mL processed AMG suspension has enzyme activity of 28321AGU when hydrolyzing Sorghum starch. While AMG capsule (PRODUCT) hasenzyme activity of 17077 AGU when hydrolyzing Sorghum starch.

Conclusion: 1 round of concentration in specific embodiments issufficient since 1 mL of PRODUCT will be absorbed to 500 mg excipientand dried providing 17077 AGU per capsule. There was a 40% loss in AMGactivity on maize starch upon encapsulating with type B calciumexcipient.

Example 6 Processed Ultrabiologic Amyloglucosidase (AMG)/Millet StarchActivity Experiment. 31 Jul. 2017 ARO/MA

Goal: To compare between AMG activity after purifying and concentrating(decrease volume 50%) suspension prepared under new protocol, describedelsewhere, and 500 mg AMG capsules activity, 1 ml of the AMG suspensionmentioned above to 500 mg of excipient B, (Calcium phosphate 10 g,Calcium silicate 10 g, Calcium Carbonate 80 g).

Given: AMW of glucose is 180.156 Daltons and 1 mg=0.0055 mmol

1 AGU (of AMG) unit activity=0.2 mol glucose released/minute (0.1 μmolmaltose equivalent (starch) hydrolyzed)

Methods Summary: Mix 75 grams crude Ultrabiologic amyloglucosidasepowder (Lot #5220511016, Exp 2.2016, BCM #700126437b) in milliQ water(qs to 250 mL). Mix well using blender (45′), then spin out crudematter, coarse and fine vacuum filter (Whatman #1, and Millipore 0.45 μMwith Whatman 42 pre-filter), and spin concentrate to 50% volume usingMillipore Centriprep 30K MWCO. Aliquot for assay of resultant lightbrown suspension. Test for absence of deoxynivalenol (Don-V) toxin.

Timed Assay (FIG. 13 ):

A) 1.00 mL AMG: 99.00 mL Millet starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

B) 1 AMG capsule: 99.00 mL Millet starch (20 mg/mL) to deliver 5.0 μL toAnalox GM9 glucometer. Measure and record starting glucose concentration(mg/dL) and during 1, 2.5, 5 and 10 minute elapsed reaction time.

Data: Don-V toxin NEGATIVE

A) For 1 ml AMG suspension:

-   -   Δ Glucose increase at 1 minute=+55.1 mg/dL

Δ Glucose increase at 2.5 minutes=+58.0 mg/dL

-   -   Δ Glucose increase at 5 minutes=+59.0 mg/dL    -   Δ Glucose increase at 10 minutes=+56.5 mg/dL

B) For 1 AMG Capsule:

Δ Glucose increase at 1 minute=+25.2 mg/dL

Δ Glucose increase at 2.5 minutes=+41.3 mg/dL

-   -   Δ Glucose increase at 5 minutes=+51.8 mg/dL

Δ Glucose increase at 10 minutes=+57.5 mg/dL

Calculations: A) For AMG suspension:

Mean 1 minute Δ Glucose increase at 5 minutes+11.8 mg/dL equivalent for+118 mg/L

1 mL AMG liberated+118 mg/min for 0.6549 mmol/min

0.6549 mmol×1000=654.9 μmol

Conversion of μmol to AGU: 654.9 μmol glucose/0.2 (for maltoseequivalents)/0.1=3274 AGU

B) For AMG Capsule:

MEAN 1 minute Δ glucose increase at 5 minutes+11.44 mg/dL equivalent for+114.4 mg/L

1 mL AMG liberated+114.4 mg/min for 0.6349 mmol/min

0.6349 mmol×1000=634.9 μmol

Conversion of mol to AGU: 634.9 μmol glucose/0.2 (for maltoseequivalents)/0.1=3174 AGU

Discussion: 1 mL processed AMG suspension has enzyme activity of 3274AGU when hydrolyzing Sorghum starch. While AMG capsule (PRODUCT) hasenzyme activity of 3174 AGU when hydrolyzing Sorghum starch.

Conclusion: 1 round of concentration in specific embodiments issufficient since 1 mL of PRODUCT will be absorbed to 500 mg excipientand dried providing 3174 AGU per capsule. There was a 4% loss in AMGactivity on millet starch upon encapsulating with type B calciumexcipient.

Example 7 Considerations

Several commercial and crude amyloglucosidase supplement products weretested and were found to significantly vary in quality. Most wereinsufficient in activity to be clinically useful and candidate fordevelopment would require enrichment by concentration with a goal of1000 AGU or more per capsule, and it is conceivable that 10 k AGU mightbe required to observe a favorable clinical response.

The data presented herein are informative and two candidateamyloglucosidase preparations were identified for potential developmentinto a medicinal food product or nutriceutical. The enzyme is heatstable and suitable for use in arid climates. AMG has a broad range ofactivity from pH 4-8 and is robust in trypsin and chymotrypsin; it wouldbe useful as a nutriceutical or supplement to relieve symptoms of starchmaldigestion. Furthermore, these products can be further processed ascapsules to be used in the field to improve the nutritional availabilityof grains that are not calorically dense or difficult to digest due torecent marasmus-like illness.

Sample 26424B was useful because of high purity, high proteinconcentration and high enzymatic activity across all tested substrates.It was known from initial work performed under standard USP assayconditions on 1% maltose (KMH31OCT14: 46-49, that AMG sample 26424B hasexcellent activity compared to Sigma-Aldrich reference standard AMG(˜41AGU per 15 mg. protein (˜3 AGU/mg AMG). The inventor originallyfound an amyloglucosidase product that has an activity that approximatesthe predicted activity of 300 IU for pure HPLC-grade enzyme. Sample26452 demonstrated activities of 286 IU for maltose, 262 IU for ricestarch and 268 IU for corn starch. Crude product approximated 50 percentprotein content, and SDS-PAGE demonstrated relatively pure protein bands(3) before and after dialysis at 50 KD MWCO and was consistent withexpected product. Bands were identical to the Sigma HPLC reference gradeamyloglucosidase (FIG. 1 ). Thin-layer chromatographic screening (FIG. 3) revealed typical residual LMW contaminants that should be easilyremoved by gradient gel filtration or dialysis during final processing.

In one embodiment, one can utilize amyloglucosidase, distributed by NEC,as a good choice for further development because it is readily availableand met minimal specifications for activity and purity. The quality israted as very good, overall, and in some embodiments is a good secondchoice for development if there was not a concern about the presence ofdeoxynivalenol (see FIG. 3 , columns 5 & 6) and if the product could bereliably delivered in active form. In other cases the Canadian supplier(Ultrabiologics, LTD) claims to manufacture a crude amyloglucosidaseproduct that exceeds 1000 AGU/gram of raw powder, but we found it toapproximate ˜3000-4000 AGU/gram raw powder with excipient.

In summary, in some cases one can utilize the methods of the disclosureon subjects with congenital sucrase isomaltase deficiency syndrome todetermine if symptoms and 13C-strach breath test improved withtreatment. This can be done under the existing BCM IRB Human Studiesprotocol, H-19253. If this is true, a field trial should be undertakento determine if amyloglucosidase, a GRAS-listed food supplement, couldimprove the nutritional status of undernourished young children.

Example 8 Sampling Labeling and Determination of Protein Concentrationby Lawry Method

Protein content of a sample may be determined by a colorimetric assaycommonly known as the Lowry Procedure for Protein Assay [Lowry, O. H.,N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Proteinmeasurement with the Folin-Phenol reagents. J. Biol. Chem. 193:265-275]. The basic principle of the test relies on complete digestionand hydrolysis of protein, peptides and amino acids to produce urea. Theintermediate, urea, is subsequently reduced with heat to form biuret(2-Imidodicarbonic diamide; H2NC(O)NHC(O)NH2) and alkaline ammonium ionthat is free to react with cupric ion (Cu [II]) to produce a violetcolor, which is then measured colorimetrically and compared withstandards to determine protein content. Under alkaline conditions (˜pH10) and the subsequent reduction of the Folin-Ciocalteayphosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by thecopper-catalyzed oxidation of aromatic acids [Dunn, M. J., 1992. Proteindetermination of total protein concentration. Harris, E. L. V., Angal,S., [Eds], Protein Purification Methods, Oxford: IRL Press]. The Lowrymethod is sensitive up to 2 mg of protein per mL. Price [1996. Proteins,Labfax, Oxford: Academic Press] notes that ammonium ions, zwitterionicbuffers, nonionic buffers and thiol compounds may interfere with thereaction, and these compounds may need to be removed or diluted beforeperforming the assays. Similar to Lowry reaction protein assay, kit isproduced by BioRad as detergent compatible protein assay kits and isavailable that uses a colorimetric method to determine proteinconcentration following detergent solubilization. DC Protein assay kitcatalog #500-0112 (Detergent-compatible colorimetric assay kit).

BioRad also produces a simple one step protein assay kit for measuringtotal protein concentration using a dye-binding method based on theBradford assay which is based upon amino acid composition. Quick StartBradford Protein assay Kit catalog #500-0202 can be adopted to use amicroplate reader. However, many detergent and basic protein buffersinterfere with the Bradford assay. Interference may be caused bychemical-protein or chemical dye interactions. The Bradford assay isbased on an absorbance shift of the dye Coomassie Brilliant Blue G-250in which, under acidic conditions, the red form of the dye is convertedinto its bluer form to bind to the protein being assayed. The bound formof the dye has an absorption spectrum maximum historically held to be at595 nm. The cationic (unbound) forms are green or red. The binding ofthe dye to the protein stabilizes the blue anionic form. The increase ofabsorbance at 595 nm is proportional to the amount of bound dye, andthus to the amount (concentration) of protein present in the sample.(Bradford M. M. Anal. Biochem. 72: 248-254, 1976.) A major problem withthis method is non-linearity over varying concentration ranges.)

Purpose

The purpose of this Standard Operating Procedure is to determine thetotal protein content in a provided candidate digestive enzyme sample.

Test Article Description and Risk Category

Description: a proteinaceous substance obtained from a GRAS-listed foodproduct or supplement or non-pathogenic organism. NONHAZARDOUS and maybe suitable for human consumption.

Frequency

Samples are prepared and analyzed on an As Needed basis when requested.

Materials and Reagents

Bio-Rad DC Protein Assay Kit (#500-0112); Detergent-compatiblecolorimetric assay kit contains: Reagent package (catalog number500-0116) includes: 250 ml REAGENT A, an alkaline copper tartratesolution 2000 ml REAGENT B, a dilute Folin Reagent 5 ml REAGENT S(Sufficient for 500 standard assays or 10,000 microplate assays) Thereagent package may be purchased as a kit with a bovine gamma globulinstandard (kit catalog number 500-0111) or bovine serum albumin standardRoche Applied Bioscience Protease Inhibitor Cocktail Tablets 20 tabletsindividually packed in foil blister packs, each tablet is sufficient fora volume of 50 ml extraction solution (04693116001)

Procedure

Summary from Bio-Rad DC Protein Assay Kit

BioRad Quick Start BSA Standard Set (set of 7 BSA standards 0.125 mg/mlto 2 mg/ml).

Run triplicate determination for all samples. Preparation of workingreagent; Add 20 μl of reagent S to each ml of reagent A that will beneeded for the run; (This working reagent A′ is stable for one week eventhough a precipitate will form after one day. If precipitate forms, warmthe solution and vortex. Do not pipet the undissolved precipitate, asthis will likely plug the tip of the pipet, thereby altering the volumeof reagent that is added to the sample.). If samples do not containdetergent, step #1 may be omitted and simply use reagent A as supplied.Use 5 dilutions of a protein standards containing from 0.25 mg/mi toabout 1.5 mg/ml protein. A standard curve should be prepared each timethe assay is performed. For best results, the standards should always beprepared in the same buffer as the sample. Pipet 20 μl of standards andsamples into clean, dry test tubes. Add 100 μl of reagent A′ or A (seenote from step 1) into each test tube. Vortex. Add 800 μl reagent B intoeach test tube and vortex immediately. After 15 minutes, absorbance canbe read at 750 nm. The absorbance will be stable at least 1 hour. (SeeTroubleshooting Guide for recommendation on using a wavelength otherthan 750 nm.) One can use Excel software to plot standard curve toderive extrapolated protein concentration SAMPLING LABELING

Upon arrival, samples are annotated with a unique numeric barcodebeginning with 700126401 and may be truncated for subsequent derivativesamples or products using the last five digits of the bar code (e.g.,26101) and further qualified using secondary alpha annotations (e.g.,26101A, 26101B, 026101C, etc.) or alternate annotation (e.g.,26101-supernatant, or 26101-precipitate, or the like).

Sample Integrity & Assessment

If not done previously, upon arrival, samples are visually inspected andthe findings recorded. This may be followed by basic gravimetricmeasures and testing for solubility in 10 mL PBS buffer solution, up toa concentration of 50% at room temperature, and vortexed. Turbid samplesare expected to yield a precipitate after centrifugation (5000 RPM×10min). Supernatant aliquots (1.00 mL×2) are oven dried (60*C) in awatch-glass and gravimetrically compared to equal volumes of PBS buffersolution. An attempt to precipitate non-turbid samples and supernatantsamples may be performed by the addition of ammonium sulfate or PEG6000or both. The suitability of the sample may be determined by proteinconcentration and purity SDS-PAGE.

Example 9 Protein Identification by SDS-Page Methodology

Non-reduced separation of enzymes by SDS-PAGE is a common method in thecourse of substrate characterization (assessment of purity and proteinsizes). Native protein mixtures must be partially denatured by a laurylsulfate detergent, SDS, until they take on a non-hydrophobic, relativelylinear conformation to permit rapid migration within a polyacrylamidegel matrix toward an applied, opposite electrical charge. Additionally,BME can be used to reduce all disulfide bonds of cysteine residues tofree sulfhydryl groups, and heating in SDS will disrupt all intra- andintermolecular interactions within the substrate. After treatment withSDS, irrespective of native substrate charges, all enzymes/proteinsacquire a high negative charge. The way of reduced and non-reducedseparation of enzymes by SDS-PAGE electrophoresis differs only by thepresence of the beta-mercaptoethanol. Within a wide pH range, thenegatively-charged protein molecules will migrate toward theelectrically charged chamber anode at a rate inversely proportional totheir relative molecular mass. Mobile substrates are usually separatedconcomitant with standard protein markers of known mass such that thereference relationship between rate of migration (Rf) and mass can beplotted and the comparative masses of unknown enzymes/proteins may beestimated. A gel cassette, containing (4-15% or 7.5% acrylamide, shouldbe expected to permit separation of polypeptides with molecular massbetween 45 and 200 kDa.

Bromophenol blue indicator, introduced into the sample,electrophoretically travels ahead of the separating protein substratestoward the anode to cue completion; after this current is turned off toavoid sample loss due to complete electrophoretic migration. Onceseparation of the proteins has occurred (between the electrode poles),the resultant bands may be visually detected using various stainingtechniques.

After optimal migration occurs, the sample gels are removed from theplastic plates and stained with a protein-binding dye [e.g., CoomassieBrilliant Blue or Gel-Code Blue® (Pierce)]. Unbound dye is removed byrepetitive washing and the amount of residual, bound dye is proportionalto distributed protein content that appears as bands. Stained gels arephotographed or scanned and the intensity of the color in each proteinband may be measured with a recording densitometer. Bands are detectedusing a Western blotting-technique that incorporates specific antibodyto the protein and chemo-luminescent detection of specific proteins onmembrane. Alternatively, if the separated constituents are radioactive(not planned), the protein bands can be detected by radiography. When apiece of photographic film is applied over the dried slab in alight-proof cassette, X-ray film will be exposed to the light in theprotein bands. After processing, dark bands appear on the developed filmand may be quantified according to band intensity.

Purpose

The purpose of this Standard Operating Procedure is determine thespectrum of proteins and enzymes contained in a candidate digestiveenzyme sample provided by the sponsor, QOL Medical, LLC.

Test Article Description and Risk Category

Description: a proteinaceous substance obtained from a GRAS-listed foodproduct or supplement or non-pathogenic organism. NONHAZARDOUS and maybe suitable for human consumption.

Frequency

Samples will be prepared and analyzed on an As Needed basis whenrequested.

MATERIALS AND REAGENTS Micropipettes and tips (Denvile Scientific,P1096-HPS) Mini-PROTEAN Tetra cell apparatus (BIO-RAD 552BR, 30 W)Trans-blot cell apparatus (BIO-RAD 49BT, 200 W) Mini-PROTEAN TGX Precastready gels (BIO-RAD 456-1085) Precision Plus Protean All Blue Standards(BIO_RAD 161-0373) 4× Laemmli Sample Buffer (161-0747) 2-Mercaptoethanol(2-ME), SIGMA (cat #M3148) 4-15% Mini-PROTEAN® TGX™ Gel, 10 well, 30 μl#456-1083 Autoradiophy film (Dennville, E3018) TRIS buffer (Invitrogen,15504-020) Glycine (Fisher BP381-5) Gel-Code Blue® (Pierce) Sodiumdodecyl sulfate (SDS) (Sigma-Aldrich L3771) CAPS(3-(cyclohexamino)-1-propanesulfonic acid) (Sigma C2632) Immobilin-Pmembrane, PVDF, 0.45 um, (EMD Millipore IPVH00010) Various primaryenzyme (target) antibodies (Cell Signalling Technologies, Beverly,Mass., https://www.cellsignal.com/) Secondary antibody-HRP (HRPanti-rabbit or antigoat or antirabbit (Santa Cruz Biotechnology)Chemoluminescent reagents (ECL Western Blotting kit, Pierce 32106) KodakAutomat (Baylor College of Medicine DDC/MVM) Milli-Q water Staining tray(Gelcode destaines with water) Milk, powder for blotting (Sigma M7409)Albumin-bovine (Sigma A9647) Ethanol, absolute>99.5% (AAPER or Sigma459844) Tween 20 Working stock solution of test sample (annotated #-WS)Precision Plus Protein™ All Blue Standards BioRad (161-0373)

PROCEDURE If sample is not pure protein, several separate techniqueswill be required, including ammonium sulfate precipitation (˜30-40%),FPLC gel filtration, affinity chromatography, and/or ion-exchangechromatography (each is a separate process and requires separate SOPs)and is dependent upon initial sample assessment, described below.

After determination of protein concentration (SOP 14-001), sample willbe diluted with PBS buffer solution to a concentration 1-5 mg/mL andlabeled as working stock (e.g., 26401-WS or #-WS).

To a 1 mL aliquot of #-WS, Laemli sample buffer with dye (1:1) to finalconcentration approximate 1-2 mg/mL (#WS/L) with for reduced and without2-ME for nonreduced SDS-PAGE

Assemble PAGE apparatus and load Mini-PROTEAN TGX Precast ready gel with15-20 uL of (#WS/L sample and with reference markers (10 uL). Note:Protein concentration should not exceed 20 micrograms.

Apply current and run gel at ˜120V for approximately 45 min.

Disconnect power and remove gel cassette from apparatus.

Remove gel from plastic retainer sheets and place gel on staining tray

Rinse gel (gently) with Milli-Q water to remove residual SDS solvent.

Stain gel with Gel-Code Blue® for approximately 1-2 hours

Destain and rinse 3× with Milli-Q water until bands appear

Photograph gel using Mobile HD Snap Camera (Sony MHS-PM5) and storeimages for transfer to E-laboratory notebook.

Analyze results with densitometer and plot molecular weight vsmigration.

Alternative to staining (for inconclusive results) is to transferproteins to membrane for Western Blotting.

Obtain PVDF membrane and immerse in absolute ethanol for 15 sec andrinse with Milli-Q water for 2 minute.

Affix gel to membrane (face-to-face).

Insert membrane cassette into Trans-blot cell apparatus and apply 400 mAcurrent for 2.5 hours in CAPS buffer

Remove cassette and discard gel

Rinse membrane with PBS/Tween20 (0.005%) X2 to remove CAPS

Dry membrane on paper towels for ˜45 min.

Re-wet PVDF membrane with absolute ethanol C2 min. while gently shaking

Rinse PVDF membrane with Milli-Q water X1 Rinse PVDF membrane water withPBS/Tween20 solution X1

Block PVDF membrane with milk (5%)×1 hour at RT or overnight inrefrigerator

Discard milk and rinse with PBS/Tween20 solution x1

Apply primary antibody (dilute 1:1000 to 1:10000 dependent upon stockspecifications)×2 hours

Rinse with PBS/Tween20 solution X3; 5 min each

Apply secondary antibody (1:10000-1:30000 dependent upon stockspecifications) X1 hour

Rinse with PBS/Tween20 solution x1; 5 min each

Add chemo-luminescent reagents [1:1 (˜5 mL/membrane)] and incubate 2minutes

Remove excess ECL with filter paper, hold damp for not more than 25minutes in x-ray cassette

Insert x-ray film in darkroom from 1 see to 1 minute, dependent uponanticipated concentration (ad lib)

Develop x-ray film in Kodak Automat developer machine

Optical computer scan film output.

Score results and record findings for analysis

Sample Integrity & Assessment

If not done previously, upon arrival, samples will be visually inspectedand the findings recorded. This will be followed by basic gravimetricmeasures and tested for solubility in 10 mL PBS buffer solution, up to aconcentration of 50%, at room temperature, and vortexed. Turbid samplesare expected to yield a precipitate after centrifugation (5000 RPM×10mins). Supernatant aliquots (1.000 mL×2) will be oven dried (60*C) in awatch-glass and gravimetrically compared to equal volumes of PBS buffersolution. An attempt to precipitate non-turbid samples and supernatantsamples will be performed by the addition of ammonium sulfate or PEG6000or both. The suitability of the sample will be determined by proteinconcentration and purity by SDS-PAGE as described herein, and will befollowing by assessment of enzyme activity, as per protocol.

Materials Safety and Data Source (MSDS)

Unless otherwise specified, physical data of the crude sample isbelieved to be a glucohydrolase substrate that is a creamy whitehygroscopic powder, bland or slightly bitter in taste. It is produced byextraction from a GRAS listed, food grade microorganism such asAspergillus niger. Substrate may be used to hydrolyze α-D-glucosides orin the brewing of beer and in the production of bread and juices.Similar enzyme substrates have been used to hydrolyze glycogen intoglucose monomers in order to study lipid accumulation in skeletalmuscle. The test compound should be capable of hydrolyzing theα-D-(1-4), the α-D-(1-6), and the α-D-(1-3) glycosidic bonds ofoligosaccharides and serve as an extracellular enzyme to convert starchto dextrins and glucose. General Molecular Formula: [H2NCHR—(S)—COOH]nwith sulphur being a variable constituent of individual,proteinogenic/composite amino acids with a total protein content thatshould approximate 30-60 units/Mg protein (biuret).

Example 10 Screening for Impurities Using Thin Layer Chromatography

Thin Later Chromatography (TLC) is a technique used for theidentification or separation of compounds in mixtures. TLC is routinelyused for monitoring the consumption of starting material in bioreactorsand to observe for the appearance of desired products and problematiccontaminants, such as pesticides and toxins. Unwanted organic productsproduced by Aspergillus or other species may include toxic contaminantsincluding mycotoxins [(e.g., deoxynivalenol) and ochratoxins)] andrarely aflatoxins. TLC uses classic, two phase principles of extractionto accomplish the separation of compounds based upon inherent compoundsolubility in a mixture: a high surface area stationary phase and amobile phase. The stationary phase consists of a thin layer (0.25 mmthickness) of absorbent silica on glass (Whatman K6 silica gel 60 Å;Cat. #4860-720; 10×20 cm). The mobile phase consists of a mixture ofvolatile organic solvents that evaporate after separation. In summary, asolution containing a particular solute substrate (e.g., invertase oramyloglucosidase) is applied near to the bottom edge of the glass TLCplate (the origin) and propped upright in a capped jar with the bottommargin of the plate submerged in the solvent. The ratio of absorbent tosubstrate must be high. With time, the solutes migrate upwards bycapillary action with the leading edge of the rising solvents passingover the substrate origin (aka: elution). Various compounds becomestationary at various levels according to inherent respectivesolubility. As the solvent rises approach the top of the plate, theplate is removed and slowly dried. One of the most importantconsiderations is the selection of a solvent mixture with an affinity tobe absorbed by the gel with full displacement of the solutes. Typicalabsorbents are highly polarized and have superb potential to elutecompounds of interest. Heat causes discolorations to appear wherevarious separated solutes became stationed. If necessary, portions ofthe silica gel can be selectively scraped, recovered, and analyzed tofurther determine identity. An ideal qualitative assessment would beabsence of extraneous solutes.

PURPOSE: The purpose of this Standard Operating Procedure is toqualitatively screen enzyme samples obtained from suppliers on behalf ofthe sponsor and determine if impurities, such as mycotoxins are present.The approach uses thin layer chromatography, which is frequently usedfor the identification or separation of compounds in mixtures.

Materials

Pre-Run solvent: 50 mL ethanol 10 mL butanol 30 mL water

A-Run solvent: 30 mL ethanol, 60 mL butanol, 10 mL acetic acid, 10 mLwater.

B-Run solvent: 65 mL ethyl acetate, 10 mL butanol, 15 mL pyridine, 10 mLacetic acid, 10 mL water.

Staining reagent:

7.0 g 4-aminobenzoic acid dissolved in methanol, 17.5 mL Orthophosporicacid Q.S. to 500 mL with methanol,

Hardware: Thin layer chromatography plates 20×10 cm (Whatman Cat.#4860-720), glass migration chamber (jar); convection oven, 10 μLsyringe

PROCEDURES: The thin layer chromatography was performed usingchromatograph glass plates coated with a 200 μm layer silica gel. Thesilica gel is an adsorbent that functions as the stationary phase. Theplates are prepared by removing 1 cm from the vertical edges and topedge of the plate. The remainder of the stationary phase is divided intoeven columns. Using a 10 μL syringe, 5 μL of a 15 mg/mL sample is placedabout 0.5 cm from bottom edge of a column. Once the sample is added, ithas the appearance of a small spot. The plate is left to dry beforeproceeding.

Polar solvents, cited above, are used as the mobile phase of thechromatography. A glass chamber jar with a removable lid is used for theTLC runs. When the plate is placed inside the chamber it must be withthe samples on the bottom of the plate. The samples must not becompletely immersed in the solvent when the plate is initially placed inthe chamber. When a TLC plate is ready to be run, the solvent is pouredin the chamber, the plate placed inside as vertical as possible, and thechamber covered.

Once the plate is loaded with samples and dried, it is placed inside theglass chamber with the Pre-Run solvent and covered. The solvent rises upthe stationary phase by capillary action. Once the solvent rises about 1cm up just above sample, the plate is removed and left to completelydry, about 1 hour. The pre-run is repeated a second time. Once dry, theplate is placed inside the chamber containing the A-Run solvent. Theplate is left until the solvent reaches as far as about 1 cm from top(this point is marked on the edge of the plate). The plate is removedfrom chamber, and left to dry, approximately 24 hours. Once it is drythe plate is placed in the chamber once more with the B-Run solution.The solvent is allowed to rise to the level that A-Run solvent reached(marked line). The plate is left to completely dry once more, againapproximately 24 hours. The samples are most often colorless; hence,when the runs are complete the separation cannot be seen. The plate isdeveloped by dipping it in the staining reagent and removing itimmediately. It is then placed in an oven at 120° C. for 20 minutes.Once it is removed from the oven, the TLC plate will show any separatedcompounds derived from the heterogeneous substrate mixture.

Test Article Description and Risk Category

Description: a proteinaceous substance obtained from a GRAS-listed foodproduct or supplement or non-pathogenic organism. NONHAZARDOUS and maybe suitable for human consumption. The volatile solvents are consideredHAZARDOUS (toxic, carcinogenic, and flammable) and require special careto avoid spills, inhalation and physical contact.

Frequency

Samples will be prepared and analyzed on an As Needed basis whenrequested.

Materials and Reagents

Pre-Run solvent: 50 mL ethanol 10 mL butanol 30 mL water

A-Run solvent: 30 mL ethanol, 60 mL butanol, 10 mL acetic acid, 10 mLwater.

B-Run solvent: 65 mL ethyl acetate, 10 mL butanol, 15 mL pyridine, 10 mLacetic acid, 10 mL water.

Staining reagent:

7.0 g 4-aminobenzoic acid dissolved in methanol, 17.5 mL Orthophosporicacid; Q.S. to 500 mL with methanol,

Hardware: Thin layer chromatography plates 20×10 cm (Whatman Cat.#4860-720), glass migration chamber (jar); convection oven, 10 μLsyringe

Procedure

The thin layer chromatography was performed using chromatograph glassplates coated with a 200 μm layer silica gel. The silica gel is anadsorbent that functions as the stationary phase. The plates areprepared by removing 1 cm from the vertical edges and top edge of theplate. The remainder of the stationary phase is divided into evencolumns. Using a 10 μL syringe, 5 μL of a 15 mg/mL sample was placedabout 0.5 cm from bottom edge of a column. Once the sample is added ithas an appearance of a small spot. The plate was left to dry beforeproceeding.

Polar solvents, cited above, are used as the mobile phase of thechromatography. A glass chamber jar with a removable lid is used for theTLC runs. When the plate is placed inside the chamber it must be withthe samples on the bottom of the plate. The samples must not becompletely immersed in the solvent when the plate is initially placed inthe chamber. When a TLC plate is ready to be run, the solvent is pouredin the chamber, the plate placed inside as vertical as possible and thechamber is covered. Once the plate is loaded with samples and dried, itis placed inside the glass chamber with the Pre-Run solvent and covered.The solvent rises up the stationary phase by capillary action. Once thesolvent rises about 1 cm up just above sample the plate is removed andleft to completely dry, about 1 hour. The pre-run is repeated a secondtime. Once dry, the plate is placed inside the chamber containing theA-Run solvent. The plate is left until the solvent reaches as far asabout 1 cm from top (this point is marked on the edge of the plate). Theplate is removed from chamber and left to dry, approximately 24 hours.Once it is dry, the plate is placed in the chamber once more with theB-Run solution. The solvent is allowed to rise to the level that A-Runsolvent reached (marked line). The plate is left to completely dry oncemore, again for approximately 24 hours. The samples are most oftencolorless, hence, when the runs are complete the separation cannot beseen. The plate is developed by dipping it in the staining reagent andremoving it immediately. It is then placed in an oven at 120° C. for 20minutes. Once it is removed from the oven the TLC plate will show anyseparated compounds derived from the heterogeneous substrate mixture.

Example 11 Estimation of Enzyme Activity by Rate of Glucose Production

There is a recognized need to supplement the diet of certain individualswith enterically-active starch digestive enzymes when inherent digestivecapacity is limited or absent. Beginning in the proximal small intestineof a normal individual, free glucose is actively absorbed into the bodyby means of the luminal SGLT-1 protein receptor-transporter. Most freeglucose is released by enzymatic digestion of larger carbohydratemolecules beginning in the duodenum. The glucose uptake receptor issodium-gradient driven by extrusion of sodium at the basolateral surfaceof the enterocyte, such that apical glucose uptake is coupled toreplacement sodium uptake. As such, the affinity of SGLT1 for glucose ismarkedly reduced in the absence of Na+ and the varied affinity of SGLT1for different monosaccharides reflects its preference for specificmonosaccharide molecular configurations, all of which have variousdownstream physiological consequences that far transcend simplehydrolysis. In summary, the ingestion, digestion, and absorption ofcarbohydrates is intricately dynamic.

In health, the system is optimized, but SGLT1 is limited by twostructural requirements for monosaccharide uptake: (1) a hexose in aD-configuration, and (2) a hexose that can form a six-membered pyranosering. As such, oligosaccharides, dextrins, and starches must becompletely hydrolyzed by luminal and mucosal derived enzymes(hydrolases) to permit efficient hexose absorption. The bulk ofabsorption occurs distal to the ampula of vater from which excretedpancreatic amylase initiates starch digestion in the lumen. Themammalian physiology has evolved to be a tiered system to hydrolyzecrude starch to dextrins and oligosaccharides over the proximal lengthof the small intestine, which is then followed by relatively specifichydrolytic activity of dextrins and oligosaccharides at the jujunalmucosal level to produce free glucose, galactose, or fructose. Illealfunction acts, in part, to scavenge residual nutrients. The teleologicaltheory holds that complete digestion and absorption ensures thatnutritional needs are met; and limits dumping of fermentable substratesinto the colon that would cause adverse symptoms.

In clinical cases, where sufficient mucosal enzymes are lacking, oralreplacement therapy is needed. This treatment approach has beensuccessfully used to hydrolyze lactose to glucose and galactose and tohydrolyze sucrose to glucose and fructose, but therapy to exogenouslyassist starchy oligosaccharide and dextrin digestion is lacking. Thework described herein relates to the quality (activity) of theparticular food-grade, fungal-derived enzymes, such as amyloglucosidase,produced by molecular biology or microbial culture techniques to releaseglucose from large target substrates in the human stomach and duodenum.

The approach to the determination of enzyme activity is to estimate therate of glucose production from observations made on a controlledsubstrate reaction between enzyme and oligosaccharides (dextrins) orstarches. The assessment of glucose production involves the use of testenzymes to hydrolyze oligosaccharides or starches, from which indirectmeasurements of resultant free glucose are made. The traditionaltechnical approach relies upon a glucose oxidation reaction that yieldsa reactive species that effects a colorimetric change that correlateswith glucose concentration, and a more modern approach relies upon achange in oxygen ion concentration that correlates with glucoseconcentration. In the later scenario, oxygen consumption from glucoseoxidation is detected by use of a Clark-type amperometric, platinumoxygen electrode. This is the same, highly accurate method upon whichblood gas analyzers are based. Test enzymes, for which activity isundetermined, can be assayed using semi-micro quantitative analysis.Exploiting the later principle, the work described herein relates to thequality (activity) of the particular food-grade, fungal-derived enzymes,such as amyloglucosidase produced by molecular biology or microbialculture, to release glucose from target substrates in the human stomach.Amyloglucosidase has broader activity than mammalian physiologicalenzymes, appears to be a robust candidate therapeutic agent, and isprobably active in the stomach. Invertase, an enzyme used to hydrolyzedsucrose, may be assayed in a similar way. The candidate digestive enzymesamples (amyloglucosidase or invertase) as provided by the sponsor weretested. HPLC-grade analytical enzymes are used for comparative purposes.Results are reported regarding milligrams glucose produced periodically(over 60 minutes) and converted to USP activity units consistent withUSP Dietary Supplements Compendium, (the United States PharmacopoeialConvention, Inc., Rockville, Md., USP/DSC 2nd ed., 2012) Appendix V,Enzyme Assays pg. 1727-1759. One unit of glucoamylase activity isdefined as the amount of glucoamylase that will produce 0.1 μmol/min ofglucose under the conditions of the assay. Other assays rely upon samplehydrolyzes of p-nitrophenyl-α-glucopyraniside (PNGP; C12H15NO8;Molecular Weight 301.25) to p-nitrophenol (PNGP) and glucose andmeasurement of PNGP at pH 4.3 and 50° C. The electrochemical (oxygenconsumption) measurement of glucose production closely equates top-nitrophenyl since the molecule is hydrolyzed in a 1:1 ratio. Theelectrochemical technology used in these experiments is exceptionallyfast with results becoming available in less than 20 seconds aftersample injection. This timely approach prevents overestimation of enzymeactivity and is remarkably free from the interference problemsassociated with antiquated PNGP optical techniques.

In a similar way, sucrose hydrolysis is defined regarding Sumner Units(SU). This is the activity of the enzyme which, under the conditions ofthe assay, will convert 1 mg of sucrose to glucose and fructose in 5minutes. One method spectrophotometrically measures the amount ofmonosaccharides produced as a 3,5-Dinitrosalycylic Acid (DNS)acid-phenol reagent byproduct correlated to a glucose standard. Themethods used herein again measures the rate of glucose productionelectrochemically. Since result fructose is unaffected by glucoseoxidase and only the glucose moiety is detected and the results must bedoubled to reflect SU or IU.

Herein, the research question asks whether a submitted candidate testenzyme could be sufficiently active in vitro to propose for furtherin-vivo use as a therapeutic replacement for luminal deficiencyconditions. The general testing approach described herein is antecedentto potential large-scale enzyme extraction, purification andpharmaceutical applications. If preliminary results appear promising,this SOP will serve as the basis for other related testing protocols(sub-SOPs) to assess potential factors that could inhibit or potentiateenzyme activity or affect enzyme stability. Purpose

The purpose of this Standard Operating Procedure is to determine enzymeactivity associated with the total glucose produced from standardizedsubstrates, including maltose, palatinose, corn LDx, potato starch, andcorn starch. The candidate digestive enzyme sample (amyloglucosidase orsacrosidase) will be provided by the sponsor or provided locally.

Test Article Description and Risk Category

Description: a proteinaceous substance obtained from a GRAS-listed foodproduct or supplement or non-pathogenic organism. NONHAZARDOUS and maybe suitable for human consumption.

Frequency

See work flow rubric, shown below. Samples will be prepared and analyzedon an as needed basis when requested by collaborating scientists at QOLMedical and supplied in suitable form. Materials and Reagents

10 mM NaCitrate buffer, pH 4.5 (10 mM Citric acid, pH 4.5 with NaOH)

Thimersol, 5% aqueous, 200 μL added to each liter of NaCitrate buffer toprevent premature glucose bacterial consumption (noted not to interferewith reactions)

Reaction vials, borosilicate with caps, 8 mL

Analox glucose analyzer and commercial reagents (glucose oxidase)

Glucose control solutions (100 and 400 mg/dL)

Pipettes

Vial warmer set within Lindberg/Blue M shaking water bath, modelSWB1122A-1 set to 37° C. (Serial number R02L-524123-RL).

Substrate A: 1% maltose in 10 mM sodium citrate buffer, pH 4.5

Substrate B: 1% palatinose in 10 mM sodium citrate buffer, pH 4.5

Substrate C: 1% corn LDx in 10 mM sodium citrate buffer, pH 4.5

Substrate D: 1% potato starch in 10 mM sodium citrate buffer, pH 4.5*

Substrate E: 1% corn starch in 10 mM sodium citrate buffer, pH 4.5**Solubilized by cooking 2% slurry for 10 minutes, bring to boil, thensimmer, then cool. Dilute 1:1 prior to use using 10 mM sodium citratebuffer, pH 4.5 Test article: as supplied.

Procedure

Prepare samples and set up test and control vials in duplicate in cold(wet ice or cold block). Note: stagger sample preps, starting each testvial at 30 sec or 1 minute intervals between measurements. e.g., 20samples+2 glucose standards in 10 minutes in the initial cycles.

In duplicate, add 2 mL stock substrate-solution (either A, B, C, D, E)into paired borosilicate vials with caps for 10 time points: 0, 10, 20,30, 40, 50, 60, 70, 80, 90 minute samples, a blank with citrate bufferonly and glucose control samples (100 μL: 100 mg/dL; 10 μL: 400 mg/dL)(#26 total). Additional glucose control samples at lower concentrationsmay be used as necessary.

Prepare 10 mL stock solution of test enzyme or reference enzyme (e.g.,1.0 mg/mL for reference A. niger sp. AMG, Sigma A7420). Use 50 mLconical tubes. The target enzyme concentration to use in this protocolis 1.0 mg per mL, as determined from previous protein concentrationdeterminations. As such, enzyme volumes will need to be adjustedaccordingly after final protein concentration is determined.

For example, samples, aliquot 0.10 mL (˜0.07) mL from stock and q.s to10 mL with sodium citrate buffer (protein concentration 143.7 mg/mL;x-factor 0.007×10).

For example, aliquot 50 mg (˜49.0 mg) from stock (core proteinconcentration: 20.4% dry).

For example, aliquot 13.00 mg (˜12.35) mg from stock and q.s to 10 mLwith sodium citrate buffer (core protein concentration: 81% dry).

Vortex for 1 minute each and centrifuge using table top IEC instrument(position #7 for 10 minutes (˜2500 RPM)), and transfer each supernate toa new 50 mL conical tube.

Qualitatively filter each supernatant using fine filter paper (Whatman#42), then repeat using very fine filter paper (Whatman #GF/C).

Optional: repeat filtration to remove residual microorganisms usingclosed-system suction-driven apparatus (Steriflip-GP, EMD Millipore#SCGP00525) and collect in sterile 50 mL conical tube.

Repeat protein concentration assays according to SOP #2 to permitaccurate enzyme dilutions and intragroup comparisons. Adjustproportionally with sodium citrate buffer to final concentration of 1.0mg/mL. o Measure glucose concentration of the glucose controls (25, 100and 400 mg/dL) using the Analox instrument

Measure glucose concentration of the test substrate (either: A, B, C, D,E) prior to adding enzyme test article. Repeat for duplicate vial.

Measure glucose concentration of the enzyme test article prior to mixingwith substrate (either: A, B, C, D, E).

Add 200 μL normalized enzyme test article to 2.000 mL substrate (either:A, B, C, D, or E). Repeat for duplicate vial.

Incubate duplicate vials at 37° C. in gently shaking water bath.

Remove 10 μL at 10 minute intervals and determine glucose levels. Repeatfor duplicate vial. Consider staggering timing for vials to account fortime taken to assay samples.

Measure glucose controls (25, 100 and 500 mg/dL) after each sample set.

Tabulate and plot all results. For each enzyme dilution, plot glucose(mg/dl) generated vs time.

Calculate glucose production rate in mg/dL/min. Select peak glucoseconcentration (e.g., 70 minute sample) and determine rise in glucoseconcentration during previous 60 minutes (e.g., [70 min mg/dL glucose-70min mg/dL glucose] and divide by 60 and record glucose production ratein mg/dL/min.

Repeat entire procedure, but increase test article 2× by adding 400 μLnormalized enzyme test article to 2.000 mL substrate (either: A, B, C,D, E). Repeat for duplicate vial.

Repeat entire procedure, but increase test article 4× by adding 800 μLnormalized enzyme test article to 2.000 mL substrate (either: A, B, C,D, E). Repeat for duplicate vial.

Glucose Standards may be 100 mg/dL Glucose Standard. Substrate may be400 mg/dL. The time duration may be 0, 10, 20, 30, 40, 50, 60, 70, 80,or 90 minutes mg/dL, for example, where measured glucose concentrationusing Analox GM9]

Substrate A: 1% maltose in 10 mM sodium citrate buffer, pH 4.5

Substrate B: 1% corn starch in 10 mM sodium citrate buffer, pH 4.5

Substrate C: 1% corn LDx in 10 mM sodium citrate buffer, pH 4.5

Substrate D: 1% potato starch in 10 mM sodium citrate buffer, pH 4.5

Substrate E: 1% corn starch in 10 mM sodium citrate buffer, pH 4.5 10.

Other Background Information

In agriculture, starch is the primary energy reserve and is found incereal seed, tubers, legumes, fruits, and vegetative tissue and provides˜70% of the calories consumed by humans. In nature, starch occurs asgranules, and the rate of enzymatic hydrolysis of these granules beforeand after physical, thermal, or chemical damage has a major impact onthe nutritive properties of the grains. From an analytical viewpoint,properties such as total concentration of starch, degree offracturization upon milling, and the extent of extent of gelatinizationdictate methodology. From a nutritional point of view, resistance todigestion in the small intestine is an important factor in determiningsuitability as foodstuffs.

In the intestine, dietary starches must be reduced to soluble dextrinsand oligosaccharides by pancreatic amylases and, in turn, the dextrinsand oligosaccharides must be reduced to monosaccharides by isomaltaseand maltase before receptor-mediated glucose uptake occurs. (Note: theprimary purpose of pancreatic alpha amylase activity is to reduce andsolubilize megamolecules to highly soluble substrates that can be easilyacted upon by hydrolytic enzymes originating at the mucosal level).Enzyme deficiency or insufficiency at either the pancreatic or mucosallevel results in failure to completely hydrolyze glycosidic linkagesthat results in maldigestion, secondary malabsorption and distal colonicfermentation. Malabsorbed oligosaccharides have been associated with thegastrointestinal distress symptoms that include pain, bloating, anddiarrhea. As such, insufficient endogenous enzyme production couldbenefit from exogenous replacement therapy, but the qualities ofcandidate replacement therapies must be known in order to determineproper substrate targeting and dosing. While pancreatic replacementtherapy has been available for decades, mucosal enzyme supplementationhas been limited to targeting lactose (with lactase) and sucrose (withsacrosidase). Conventional medicine has largely ignored maldigestiveissues related to the bulk of the dietary carbohydrate load(“starches”), and OTC remedies are not known to be truly efficacious.

The most important feature of mucosal enzyme activity (efficiency) isproduction of free glucose from its immediate and primaryoligosaccharide substrate and activity is reported as a rate of glucoseproduction in terms of milligrams glucose per gram of substrate per unitof time (minutes). Other features, such as physical characteristics andstability, are addressed elsewhere. The technical measurement ofpolysaccharides or oligosaccharides in food substrates involvesdegradation to the reducing sugar components (glucose), which then canbe measured either enzymatically and coupled with colorimetry, or byemploying various instrumental procedures such as high-performanceliquid chromatography (HPLC). A useful method is faster and uses theprinciple of electrode detection of enzymatic oxygen uptake by theoxidoreductase and dehydrogenase enzyme-mediated glucose reaction.Starch or soluble oligosaccharides [limit-dextrins (LDx)] concentrationscan be expressed in terms of dextrose-equivalents orglucose-equivalents. The later electrochemical glucose detection methodrelies up measurement of oxygen consumption from the production ofhydrogen peroxide by glucose oxidase and, under the conditions of theassay the rate of oxygen consumption is directly proportional to glucoseconcentration. This assay approach is accurately detected by anoxygen-sensing electrode (ANALOX, London, England, PMID: 7762948) and isbelieved to lessen the chance of technical errors. Final results aredetermined by regression analyses against standards.

Limit dextrins (LDx) are oligosaccharides that are incompletelyhydrolyzed only to the limit that pancreatic alpha amylase willfacilitate the reaction to progress. LDx is the transitional nutritionalsubstrate that is produced in the intestinal lumen. Complete digestionrequires further hydrolysis at the mucosal level before absorption canoccur. As such, insufficient endogenous enzyme production requiresexogenous replacement therapy, and the qualities of candidatereplacement therapies must be known to determine proper dosing. As such,maltose, LDx, palatinose (as a surrogate for isomaltose) and cookedstarch are the target substrates that this work is most concernedbecause they represents the natural (luminal) post-pancreatic dietarysubstrates that are produced for luminal-mucosal-bound hydrolases to actupon (the brush border enzymes or endo-amylases) and this is a criticalstep in the hydrolytic process when, theoretically, maldigestion can beprevented. Since LDx is highly soluble, stable, and extremely accessibleto amyloglucosidase hydrolysis, it is among the most desirablesubstrates for in vitro work. Furthermore, since corn starch is one ofthe primary dietary starches and is free of gluten, it is also apreferred species upon which to test various amyloglucosidase (AMG)activities. Potato, tapioca, and edible algal starch are also consideredgood crude sources for LDx production. The rate of LDx hydrolysis alsodepends on the type of linkage and on the chain length of the substrate,α-1-4 linkages are more easily hydrolyzed than α1-6 or α1-3 linkages.However, maltotriose and in particular, maltose and palatinose arehydrolyzed at slower rates than higher oligosaccharides, presumptivelydue to the rapid glucose production that inhibits AMG activity. As such,it is unlikely that reactions could go to absolute completion. Todevelop test parameters, several typical reaction analyses are shownbelow.

A prototype assay was developed using commercial, referencereagent-grade AMG (Sigma-Aldrich A9229) diluted to a concentration of 2mg/mL (100 uL AMG) and provided a model for a standard reaction andwhich produced free glucose at a rate of 0.2 mg/dL/hour (1.4 uM/min).

It was concluded that an enzyme concentration of 1 mg/mL would besufficient for future work. A second pilot test using maltose as asubstrate was performed on 24 Jun. 2014 using 100 uL of 1 mg/mLamyloglucosidase (Sigma-Aldrich A9229) stock solution in 1 mL of 10mg/mL maltose (Sigma-Aldrich M2250) substrate stock solution.

The expectation was that robust hydrolysis would occur, sinceamyloglucosidase is selective of alpha 1-4 linkages. The beginningglucose content of 1 mg/mL (100 mg/dL) amyloglucosidase stock solutionwas 8.1 mg/dL and the beginning glucose content of 10 mg/mL (1000 mg/dL)maltose stock solution: 9.5 mg/dL. The resultant data curve demonstratedan excellent free glucose production rate of 9.2 mg/dL/min.

Example 12 Use of Amyloglucosidase Supplementation in Functional BowelDisorders, Congenital Sucrase Isomaltase, Small Bowel Mucositis, andProtein Calorie (Energy) Malnutrition

In health, the intestinal mucosal cells function to absorbmonosaccharides from hydrolyzed complex dietary carbohydrates and, whencells are damaged (illness), they are significantly impaired fromcompletely hydrolyzing oligosaccharides and this leads togastrointestinal symptoms. The mucosal cells are dynamic and assimilatethe luminal digestive products and transfer nutrients to variousendogenous metabolic processes (Ravich and Bayless, 1983). At theearliest level, food digestion critically depends upon secretion ofsalivary and pancreatic α-amylase; then is completed by cellularexpression of several other apical digestive enzymes. First, dietarystarch is ‘coarsely’ hydrolyzed in the duodenum and jejunum, byintraluminal alpha-amylase to release soluble maltose, maltotriose,oligosaccharides and alpha-limit dextrins. These intermediateby-products of partial digestion are not directly absorbable and mustundergo further enzymatic hydrolysis (cleavage) at the apical enterocytesurface to monosaccharides (glucose, fructose, and galactose) byexpressed sucrase-isomaltase (SI), maltase-glucoamylase (MGAM), andlactase. These enzymes, along with trehalase (a minor player), arecontinuously produced by healthy luminal enterocytes that constitute theapical mucosal surface, also referred to as the brush border because ofthe exposed velveteen-appearing microvilli. Released monosaccharides,mostly free glucose, enter the absorbing enterocyte at the apicalsurface by specific transport mechanisms (“carriers”) and aredistributed to the body using and inhibit hepatic gluconeogenesis. It iswell known that mucosal enzyme activity is frequently reduced in statesof inflammation, functional bowel disorders (Sinagra et al., 2016),malnutrition (Mehra et al., 1994), and mucosal injury (Stringer et al.,2007), and this is a largely unaddressed therapeutic concern.

The complete digestion of starch is thought to be predominantly relianton the proper expression and function of the sucrase-isomaltase (SI)enzyme complex [chromosome 3q26.1](Auricchio et al., 1963) anddeficiency conditions exist. The SI is a complex composed of twoα-glucosidase units, sucrase, and isomaltase (Conklin et al., 1975). Theisomaltase component demonstrates considerable α-glycosidic activity onstarch-derived glucose oligomers, and the SI complex togethercontributes to 60 to 80 percent of total intestinal maltase activity(Gericke et al., 2016). The isomaltase component of the SI complexhydrolyzes 1,6 linkages of the α-limit dextrins after primary luminalpancreatic amylase has reduced the ingested starch (Galand, 1989).Surprisingly, its role in causing symptoms of maldigestion has beenlargely ignored until recently when our group made advances in thefield. Secondarily, mucosal maltase-glucoamylase (MGAM), a chromosome7q34 gene product, is relatively restricted to hydrolyze alpha 1-4 bondsof soluble oligosaccharides, and the SI component works in dominantconcert to hydrolyze α-1-6 bonds and complete digestion to glucose(Diaz-Sotomayor et al., 2013). MGAM is noted to have a significantamount of compositional amino acid sequence homology, but isinsufficient to make up for the loss of SI. Acquired and congenitalenzyme deficiency syndromes can be due to specific gene mutations (Genget al., 2014) and secondary transport errors or combinations thereof(Jacob et al., 2000; Puntis, 2015). There are needs for an AMGsupplement or medicinal food to assist those with irritable bowelsyndrome and malnutrition associated with several etiologies.

Using crude AMG purchased from UltraBiologics (GRAS), in MontrealCanada, the feasibility of using AMG taken by mouth to aid humandigestion of starches was successful. The aim was to provide 16-thousandactivity units (AGU) per capsule; which is in line with current dosagestrengths for α-amylase contained in Pancrease HL® USP. AMG is sourcedfrom the fermentation of carbohydrates by Aspergillus niger, is crude(possibly derived from the elution of the mash). Crude AMG thought tocontain various aberrant substances, not limited to aliphatics,aromatics and possibly trace amounts of organic mycotoxins, such asdeoxynivalenol (aka: vomit toxin) (Bennett, 2003). A critical featurethat was exploited for purification purposes is that AMG is completelyinsoluble in absolute ethanol. As such, a technique to prepare highstrength AMG capsules without aberrant substances has been outlinedusing semi-micro quantitative analyses (e.g., enzyme activity) and thinlayer chromatography (TLC). Newly produced capsules were taken by mouthand were well tolerated with adverse events. Efficacy studies areunderway (IRB Approved Protocol H-19253) using a modified stableisotope-labeled tracer technique (Opekun et al., 2016).

Several experiments were performed to compare the activity of purifiedAMG compounded with a powder excipient (B) containing 80% calciumcarbonate+10% Calcium phosphate+10% Calcium silicate. The Excipient Bresults showed minor 45%, 35%, 73%, 45% and 4% decreases in the enzymeactivity on sorghum, rice, wheat, maize, and millet respectively. Theseexperiments need to be confirmed. Then another set of experiment wasdone to define whether concentrating the AMG enzyme using acentrifugation technique (Centriprep®) or overnight evaporationtechnique resulted in obtaining a higher enzyme activity compared tonon-concentrated AMG. The results showed the 2×AMG, from overnightevaporation concentration technique, carries the highest enzymeactivity, even higher than the 4×AMG from Centriprep® concentrationtechnique. The aforementioned experiments demonstrated excellent ratesof glucose production approaches a steady state level after 5 minutes.An additional experiment was done by diluting the AMG enzyme andincreasing the volume of the sorghum substrate to mitigate inhibition ofactivity. The anticipated results of that experiment indicate that highglucose levels inhibit activity, which is a desirable outcome ofclinical utility. Comparing different technique of AMG capsulespreparation; using excipient A (80% calcium phosphate, 10% Calciumcarbonate and 10% Calcium silicate) along with the 2×AMG from overnightevaporation concentration, excipient A has the highest enzyme activity(19K AGU) and the shortest preparation (drying) time.

The present disclosure provides a significant advance in the developmentof AMG and desire to partner with GMP industry to produce and market ahigh potency AMG nutritional supplement or medicinal food. The essentialsteps to produce potent AMG capsules have been worked out and includesourcing of good quality crude AMG, purifying it (washing) it from atoxins and irritants with absolute alcohol, removing insolubleparticulate matter (mash) derived from the fermentation process, mixingthe aqueous product with excipient, air drying, encapsulation (#00),packaging, labelling and distribution for sale. Such a product is inhigh demand, especially from among those who suffer from irritable bowelsyndrome (˜30% of the U.S. population) and garner great interest fromparties that are addressing the issue of world hunger.

Example 13 Example of AMG Purification Method

One specific example of an AMG purification method is as follows:

1) Verify crude stocks of amyloglucosidase and QA, Chain of Custody fromUltraBiologics [UB Lot #1109171347, BCM #700126464]

2) 1 part CRUDE AMG (UltraBiologics): 1 parts Absolute ETOH

60 grams AMG=20 mL dry volume dispersed as 20 mL per 50 mL conical tube(#4) and to each was added 20 mL 100% ETOH and 10 μlass beads. Each wasvortexed 5 minutes. Glass beads were removed.

3) Swing arms centrifuge (mushroom) at 2500 RPM for 30 minutes, aspirateall ETOH and extracted toxic lipid top layer to waste [note that toxiclipids were obvious (5+)]

4) Repeat steps 2 and 3, once. [note that supernatant showed minimalpresence (1+) of lipids for QA].

5) Retain solids, q.s. 50 mL conical each with Milli-Q water, combine toa beaker (200 mL) and hand blend for 5 minutes (Kitchen Aid KHB1231).

7) Centrifuge at 2500 RPM for 45 minutes, recover (decant) supernatantwith AMG, discard sediment ppt.

9) Buchner suction filter using Whatman #2 retains>8 uM under lowsuction.

10) Fixed arm centrifuge 6000 RPM for 59 minutes to recover clear tansupernatant (˜40 mL). N.b., this essentially concentrates the finalproduct by altering the final ratio of product to excipient and preventssubsequent clogging of the adsorptive sprayer. (Yield approximates, 60mL, qualitative tests POSITIVE for protein ˜50 mg/dL).

11) Adsorb to excipient (calcium carbonate, calcium phosphate, sodiumsilicate 8:1:1) in 2:1 ratio AMG:excipient.

12) Dry overnight on stretch plastic wrap tray under Class 100 blowinghood and UV light.

13) Collect product as flakes and chunks to clean high-speed coffeegrinder.

14) Sift to clean tray, repeat pulverization on any retained particlesand transfer to 50 mL conical.

15) Suspend in absolute ETOH 1:1 and vortex 5 minutes.

16) Centrifuge at 2500 RPM for 15 minutes, recover sediment ppt, discardalcohol (should be crystal clear, no lipid layer).

17) Disperse solid to stretch plastic wrap tray under Class 100 blowinghood and UV light for 2 hours (spread thin to dry quickly).

18) Encapsulate to #00 capsules (750 mg each). [fine light tan powder,homogeneous]

19) Label as Lot #AMG ddmmmyy

19) Test for deoxynalevenol (DON-V, Vicam Millipore). The specificationshould test negative to less than 0.05 ppm.

20) Test for AMG starch hydrolysis activity as per SOP. Thespecification should exceed 7500 AGU per capsule.

Example 14 Development of AMG Capsules for Human Consumption

Disaccharidase deficiency syndrome results in the inability to absorbingested saccharides in the gastrointestinal tract because of thefailure to completely hydrolyze saccharides within the lumen of thesmall intestines. Complex sugars (oligosaccharides) and starches arenormally metabolized to monosaccharides by the intestinal enzymesmaltase-glucoamylase and sucrase-isomaltase and absorbed in the smallintestine. Failure to hydrolyze oligosaccharides and starch(maldigestion) to monomers results in symptoms of gastrointestinalmalabsorption. As a prototypical condition, congenitalsucrase-isomaltase insufficiency disease (CSID) is a rare, geneticdisorder that results in reduced sucrase-isomaltase (SI) enzymaticactivity necessary for cleaving the disaccharide sucrose and starch inthe small intestine. Acquired deficiency conditions also exist.Nevertheless, loss of SI results in adverse gastrointestinal signs andsymptoms, which may be incapacitating.

SI deficiency often leads to symptoms such as bloating, abdominal pain,and chronic diarrhea as maldigested carbohydrates pass into the largeintestine causing an osmotic load, bacterial fermentation, andaccelerated transit in a manner similar to lactose intolerance. As aresult, patients deficient in SI have reduced nutrient absorption,chronic malnutrition, or failure to thrive. Small intestinal endoscopywith mucosal biopsies is the historical “gold standard” for diagnosingSI deficiency using a disaccharidase activity assay. The enzymaticactivity level of SI are measured as a ration to protein levels found inthe biopsies and compared with normal values.

This disclosure puts forth the concept that ingestion of non-toxicfungal-derived enzymes [e.g., amyloglucosidase, (AMG)] couldsignificantly aid in the hydrolysis of dietary starches (CAS Number:9032-08-0) in the absence of effective isomaltase activity.Administration of AMG should be helpful as a nutriceutical or medicalfood to release glucose in the stomach, with or without the addition ofinvertase, which hydrolyzes sucrose (table sugar) to glucose andfructose.

Description of Test AMG Article: Purified amyloglucosidase in Milli-Qwater adsorbed to inert excipients (Calcium phosphate 10 g, CalciumSilicate 10 g, Calcium Carbonate 80 g or various proportions thereof)and dried. The materials are GRAS, considered NONHAZARDOUS and aresuitable for human consumption without regulation. Minimal risk.

Examples of Procedures

The counter, analytical balance, instruments, and vent hood must becleared, clean with warm/soapy water followed by ethanol wipe down.Working area and Class 100 clean bench hood must be lined with newplastic lined absorbent pads. Personnel should don disposable nitrilegloves, protective garments, eye protection and disposable face maskwhen preparing product for human consumption. All disposables should befood-grade safe clean or sterile and non-disposables should bedeionized-filtered water rinsed, acetic acid rinsed (1%) and air driedin safe space covered with protective shield until ready for use.Working area is a spark-free zone.

Amyloglucosidase Preparation: Measure 75 g crude AMG (Lot: 5220211016),place in a clean 1.2 L chilled blender jar (with cap), admix 150 mL coldabsolute ethanol, USP and emulsify using a high speed commercial blender(Waring 700, Model 31BL46, New Hartford Conn.) for 15 minutes, takingcare to avoid excessive heat. (Total volume should approximate 240 mL.)Equally aliquot the resultant AMG/ethanol suspension by aliquoting ˜40mL of suspension to six 50 mL conical tubes (Corning). Centrifuge theconical tubes with AMG solution at 6,000 rpm for 60 minutes usingfixed-arm centrifuge at 10 degrees Celsius; after centrifugation, threelayers will be observed. Using disposable Pasteur pipettes or vacuumaspiration apparatus, aspirate the floating top lipid/debris layer towaste, decant the ethanol solute from each conical tube and holdsupernatant for assay of deoxynivalenol. Retain the precipitatecontaining AMG in each conical tube. Label as indicated.

Using each precipitate, from above, repeat the ethanol wash process withminor variations by adding 10 grams of clean glass or stainless steelmixing balls to each conical tube, admix cold absolute ethanol, USP(q.s. 50 mL each conical tube) and vigorously vortex for 5 minutes.Centrifuge each AMG solution again at 6,000 rpm for 60 minutes, decantthe ethanol solute and retain both the precipitate and supernatant fortesting. This ethanol wash step maybe repeated if it is determined thatexcessive amounts of deoxynivalenol persists according tospecifications.

Extract the AMG from the sediment by admixing cold purified water (e.g.,3M Water Dual Port Water Filtration System—. DP290, 0.2 Micron Ratingand 10 GPM) (q.s. 50 mL each conical tube or a ˜2:1 ratio water to AMG)and vigorously vortex for 5 minutes. Centrifuge each AMG solution at6,000 rpm for 60 minutes. Pour off the AMG solution/sediment suspensionto vacuum filtration apparatus. Using a modified filter flask, vacuumline, and Whatman-GE GF8 glass filter (Cat. #10370105), transfer thecontents of each conical tube to filter receptacle and collect AMGsolution and discard sediment. Repeat filtration process in separateapparatus using Whatman-GE #2 qualitative filter, aliquot 5 mL forquality control and assay and retain volume balance for admixing toexcipient and drying.

Resultant AMG solution should be light brown and test positive forprotein in excessive of 50 mg/dL by dipstick reagent (Bayer HealthcareModel: 2184). Product glucose concentration is expected to benegligible. Total expected yield of purified product is 200 mL.

Quality Control Assays

Deoxynivalenol: determination of deoxynivalenol concentration: air dryat room temperature 10 mL aliquot of each ethanol wash solution andreconstitute using 400 uL commercially-supplied VICAM DON-V diluentsbuffer (#100000248) solution and apply 100 uL to VICAM DON-V test stripaccording to manufacturer's instruction using VICAM Vertu, Lateral FlowReader equipped with VICAM Don-V quantitative test strips and recordedwith a GeBE-FLASH, easy-load desktop-thermal printer and record resultas parts-per-million corrected for 10 mL concentrated aliquot. Note:direct assay for deoxynivalenol of crude AMG (aqueous) is expected toresult in false negative outcomes due to contaminate interferences knownto exist.

Activity: under separate SOP, measure the enzyme activity 1 ml ofrefined AMG solution, or strict portion thereof, then calculateenzymatic activity for desired starch substrate using Analox GlucoseAnalyzer (GM-9) (or suitable alternative). Typically sorghum and riceflour are used since those substrate are low in protein content.

Protein Content: Under separate SOP, determine protein concentration ofAMG (Lowry Method) if required. Resultant value needed for SDS-PAGE.

Identification: Under separate SOP, confirm presence of AMG usingSDS-PAGE compared with commercial reference standard (Amyloglucosidasefrom Aspergillus niger, Sigma-Aldrich A7420, MW 97 KD).

Amyloglucosidase Capsule Preparation: Admix 200 mL refined AMG solutionto 100 grams of mixed calcium salt excipient (USP), such carbonate (8parts), silicate (1 part) and phosphate (1 part) mixture to a cleanbeaker and mix well; note that to excipient proportions should beoptimized for mechanical capsule filling. Pour mixture to drying trayslined with food-grade plastic wrap and permit to air dry overnight inlow humidity clean room or under Class 100 hood. Collect dried mixtureand pulverize using clean blender or coffee grinder until particle sizemeets specifications suitable for standard pharmaceutical-grade gelatincapsule filling. Alternative procedures for admixing, including sprayapplication of enzyme to excipient, have been described elsewhere andmay be suitable depending upon up-scaling needs. Using resultant valuefor final product AMG activity (see above), the product should beproportionally adjusted for desired activity using neutral calcium saltexcipients to obtain desired dosage to fill ratio for either #00 or #0sized gelatin capsules. AMG activity specification may approximate 4000AG activity units or more (up to 20,000 AGU). This procedure is subjectto alterations depending upon equipment availability and currentstate-of-the-art for substrate encapsulations.

In specific embodiments, one can ingest 2 capsules by mouth per meal andthe capsules may be stored in a cool and dry place

Example 15 Amyloglucosidase Supplementation Corrects Small IntestinalIsomaltase Insufficiency in Symptomatic Patients

Carbohydrate (CHO) maldigestion due to mucosal disaccharidase deficiencyoccurs with hypolactasia and primary or secondary sucrase-isomaltase(SI) deficiency. When CHO load exceeds digestive capacity patientsdevelop symptoms of abdominal discomfort, bloating and change in stoolcharacter. Enzyme supplements are available for lactose and sucrosedigestion. However, starch and dextrins constitute more than 50% of atypical diet and supplemental digestive enzymes are not available forpost-pancreatic starch digestion (mucosal level dextrins and solubleoligosaccharides). While FODMAP restriction diets appear to be helpful,supplemental amyloglucosidase (AMG) directly addresses the cause ofsymptoms. The present example tests whether AMG supplementationaccelerates starch digestion.

METHODS: Stable isotope ¹³C-labeled starch load meals and ¹³CO₂ breathenrichment analyses were used to assess digestion and assimilation.After obtaining IRB approved and informed consent, eight subjectsunderwent paired ¹³C-starch breath testing (U.S. patent Pend. Ser. No.15/083,048; PMID 27579322) using a standardized 45 gm 150 mg13C-starch)/240 mL tracer-labeled rice pudding meal with and withoutrefined AMG supplementation (20K AGU per 2 capsules), respectively onseparate days. Changes in isotopic enrichments were measured andcompared to the non-supplemented tests. Subjects included three youngwomen with compound heterozygosity for congenital sucrase-isomaltasedeficiency (CSID: 3p25.2-26) with near zero mucosal SI disaccharidaseactivity, one heterozygous man with CSID and diminished SI activity, onesymptomatic man with IBD, one symptomatic woman with mixed IBS, and twohealthy women (asymptomatic controls) that tolerated all high-starchcontent meals without symptoms.

RESULTS: supplemental AMG resulted in increased starch digestion inthose with CSID, IBS, and IBD. Comparing area of enrichment under the180 min timed curve, there was a 57% increase in digestive oxidation inthe CSID patients (¹³CO₂ Δ over the baseline enrichment ‰; FIG. 15 ), a28% enrichment increase in the heterozygous CSID subject, and ˜32% & ˜6%enrichment increase in the two patients with IBD and IBS-M,respectively. The two healthy controls showed no improvement with AMGtreatment over inherent digestive capacity (Table 3). There were noadverse events.

TABLE 3 All subject data for 3-hour ¹³C-starch tracer labeled (150 mg)rice pudding (45 gm load) breath test response to amyloglucosidase mealsupplementation (20K AGU) in 8 subjects. Compound Compound Compound IBS-IBD- Healthy Healthy Heterozyg. Heterozyg. Heterozyg. Heterozyg. Mix UCControl Control Status CSID 255 CSID 156 CSID 155 CSID 296 C3 D4 A1 B2Gender- F (15) F (17) F (17) M (18) F (32) M (28) F (22) F (25) Aged(Years) HEIGHT 1.6 1.4 1.5 1.7 1.6 1.8 1.5 1.6 Body Mass 59.9 50.3 51.779.4 77.0 86.1 63.5 60.0 (KG) CO2 1.86 1.05 1.07 1.49 1.45 1.53 1.221.18 Production Rate mM/min Pre- 427 379 344 520 647 499 853 826treatment 3° AUC Enrichment AMG On- 731 650 439 666 685 659 782 776treatment 3° AUC Enrichment 3° delta +71.2% +71.5% +27.5% +28.1% +5.8%+32.1% (−9.1%) (−13.7%) enrichment with AMG treatment

AMG supplementation (20K AGU) improved starch digestion as measured by¹³C-tracer-labeled breath testing. AMG treatment approach may offer aneffective therapy for CHO malabsorption in CSID, IBS, IBD, andiatrogenic mucositis. If true, AMG would be the first therapy proposedto address starch maldigestion in patients with CSID. Further testing iswarranted to determine appropriate dosing to achieve maximum clinicalbenefit for the conditions cited above, to realize other applicationsand fully characterize the physiological effects.

Example 16 Amyloglucosidase Supplementation Corrects Small IntestinalIsomaltase Insufficiency in Symptomatic Patients with IBS and CongenitalSucrase-Isomaltase Deficiency (CSID)

Carbohydrate (CHO) maldigestion because of disaccharidase deficiencyoccurs with hypolactasia and primary or secondary sucrase-isomaltase(SI) deficiency. Cases of SI deficiency have been shown using stableisotope breath testing (BT), by genotyping and by Dahlqvist activityassays on performed on duodenal mucosal samples. When CHO load exceedsproximal digestive capacity, typical symptoms of abdominal discomfort,bloating and change in stool character ensue. Enzyme supplements areavailable for lactose and sucrose digestion, but not yet to completestarch digestion (˜70% of the habitual diet). α-amylase is available forpartial starch digestion to dextrins, but hydrolysis must be completedat the mucosal interface to permit glucose absorption. Oligosaccharidesthat pass to the colon are noxiously fermented. While dietaryrestriction appears to be helpful in relieving symptoms, it isdifficult; and supplemental AMG improves proximal oligosaccharidedigestion, in specific embodiments of the disclosure.

The present example characterizes whether oral AMG supplementationaccelerates starch digestion. The inventor used stable isotope¹³C-labeled starch load meals, ¹³CO₂ BT enrichment analyses and bloodglucose (BG) monitoring to assess starch digestion and assimilation.After obtaining IRB approved consent, two subjects underwent paired¹³C-starch BT (PMID 27579322) using a standardized ¹³C-labeled cornstarch porridge with and without refined AMG supplementation (20K AGUper 2 large capsules or 4000 AGU per 4 small capsules) on separate testdays. Changes in ¹³CO₂ BT enrichments and BG were measured and comparedto the non-supplemented tests. Subjects included 2 women: one withcompound heterozygosity for CSID (3p25.2-26) with near zero mucosal SIactivity and one with marked mixed-IBS symptoms.

Supplemental AMG increased starch digestion in those with CSID and IBS.Using 20K AGU dose, the area under the BT enrichment curve increased 54%(120 min) in the CSID patient (¹³CO₂ Δ over the baseline; FIG. 16 ).Using the 4K AGU dose, the area under the enrichment curve increased 55%in the IBS patient and was slightly diminished in the CSID case. Thecolumn on the left shows a robust response to 20,000 AGUamyloglucosidase in a patient with CSID; the column in the center showsa no response to 4,000 AGU amyloglucosidase in a patient with CSID andindicates that a dose-response relationship exists, and the column onthe right shows a robust response to 20,000 AGU in a patient withmixed-form irritable bowel syndrome (IBS-M). The non-response (centercolumn 4000 AGU) indicates that the response was too small to bedetected or that the released glucose was assimilated into body storesand that there was no excessive glucose available for oxidation andbreath test detection at 120 minutes ET.

Blood glucose (FIG. 17 ) is presented as change (delta) in blood glucoseconcentration response from baseline, measured in humans at 45 minuteselapsed time, to one oral dosage of purified amyloglucosidase. There isa 118%-rise at 45 min (IBS) and 77% rise at 45 min (CSID). There were noadverse events. The column-pair on the left shows a robust response to4,000 AGU amyloglucosidase in a patient with CSID; and the column-pairon the right shows a robust response to 4,000 AGU amyloglucosidase in apatient with mixed-form irritable bowel syndrome (IBS-M).

Thus, the 20K AGU AMG supplementation significantly improved starchdigestion in the case of CSID as measured by ¹³C-tracer-labeled breathtesting. The 4K AGU AMG supplementation improved starch digestion in thecase of IBS as measured by rise breath enrichment and demonstrated arise in blood glucose (both cases). In a particular embodiment, there isa dose-response relationship, and dosing may in at least some casesdepend upon meal load and habitus. AMG treatment approach offers aneffective therapy for CHO malabsorption at least in CSID IBS and otherenvironmental enteropathy. AMG is the first therapy to address starchmaldigestion in these patients.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

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Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the design as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thepresent disclosure, processes, machines, manufacture, compositions ofmatter, means, methods, or steps, presently existing or later to bedeveloped that perform substantially the same function or achievesubstantially the same result as the corresponding embodiments describedherein may be utilized according to the present disclosure. Accordingly,the appended claims are intended to include within their scope suchprocesses, machines, manufacture, compositions of matter, means,methods, or steps.

What is claimed is:
 1. A method for optimizing starch or fructansdigestion in an individual, comprising the step of providing to theindividual an effective amount of an amyloglucosidase compositionproduced by the method of extracting a crude amylglucosidase compositionin the presence of an immiscible solvent, and separating a purifiedamyloglucosidase composition from the immiscible solvent, wherein thecomposition comprises amyloglucosidase in a dose that is equal to orgreater than 10,000 unit releases of one gram of glucose per hour (AGU).2. The method of claim 1, wherein the individual is in need of treatmentor prevention of a condition selected from the group consisting ofcongenital sucrase isomaltase syndrome, functional bowel disorders,functional bowel syndrome, functional duodenal disorders, small bowelbacterial overgrowth, radiochemotherapy-induced mucositis and short-gutsyndrome.
 3. The method of claim 1, wherein the providing step occursdaily, weekly, monthly, or yearly.
 4. The method of claim 1, wherein theindividual is malnourished.
 5. The method of claim 1, wherein theindividual is an infant, child, adolescent, teenager, or adult.
 6. Themethod of claim 1, wherein the composition is formulated in a comestibleor beverage.
 7. The method of claim 6, wherein the composition is a foodsupplement.
 8. The method of claim 1, wherein the composition is in theform of a solid, liquid, or gel.
 9. The method of claim 1, wherein thecomposition is a capsule, tablet, pill, film, lozenge, powder, orcombination thereof.
 10. The method of claim 1, wherein the immisciblesolvent is absolute ethanol, acetone, Dimethyl sulfoxide, methanol, or acombination thereof.
 11. The method of claim 10, wherein the solventfrom which the purified amyloglucosidase composition is separatedcomprises one or more toxins.
 12. The method of claim 1, wherein thecrude amylglucosidase composition is in the form of a solid.
 13. Themethod of claim 1, further comprising repeating the extracting andseparating steps one or more times.
 14. The method of claim 1, furthercomprising dissolving the purified amyloglucosidase composition in waterto form a solution, and filtering the solution.